Vaccines formed by virus and antigen conjugation

ABSTRACT

Disclosed herein are methods of forming compounds and exemplary compounds in the nature of a conjugated compound, which in some embodiments comprises an antigen and virus particle mixed in a conjugation reaction to form a conjugate mixture, such that the conditions and steps of forming these products allow for use of the conjugate mixture as a vaccine, including but not limited to use as a vaccine against various pathogens including for treatment of diseases caused by novel coronaviruses (including SARS-COV 2).

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a continuation-in-part application under 35U.S.C. § 120, for which common subject matter, if any, is entitled tothe benefit of the filing date of U.S. Nonprovisional patent applicationSer. No. 16/709,063, filed on Dec. 10, 2019, which is acontinuation-in-part application that claims the benefit of and priorityto U.S. Nonprovisional patent application Ser. No. 16/437,734, filed onJun. 11, 2019, which is a utility application that claims the benefit ofand priority under 35 U.S.C. § 119(e) to U.S. Provisional PatentApplication No. 62/683,865, with a filing date of Jun. 12, 2018, andfurther under 35 U.S.C. § 119(e) priority is claimed with U.S.Provisional Patent Application No. 63/013,284, filed on Apr. 21, 2020.The teachings and entire disclosure of all aforementioned applicationsare fully incorporated herein by reference.

REFERENCE TO A SEQUENCE LISTING

This application contains a Sequence Listing in compliance with 37C.F.R. § 1.52(e) that is hereby incorporated by reference in itsentirety. The sequence listing text file submitted via EFS contains thefile “KBP_SequenceListings.txt” as a computer readable form, that wascreated on Aug. 4, 2020, and is 10,824 bytes in size.

FIELD OF INVENTION

The embodiments described herein include use of a multi-set process forproducing highly purified, recombinant viruses as antigen carriers, andstill further various embodiments relate to vaccine production using apurified virus and a purified antigen, including vaccines intended forprevention of novel coronaviruses, such as SARS-CoV2 (referred to hereinas Covid-19 or SARS-2.) Disease.

BACKGROUND

Viruses have a nucleic acid molecule in a protein coat and replicateonly inside the living cells of other organisms. Often thought of asharmful, a wide range of viruses are capable of infecting all types oflife forms such as humans, livestock, and plants. Yet on the positiveside, there is growing interest to use viruses for a range oftherapeutic purposes, including without limitation vaccine creation,gene therapy, and cancer treatments, to name a few. However, to studyviruses, understand their structure, and adapt viruses for moleculartools and for disease therapy vectors and carriers, viruses first mustbe purified to remove any cell debris, macro-molecular fibers,organelles, lipids, and other impurities that would interfere with theintended function of the virus.

Once purified, viruses are suitable for a number of uses. One that isrelevant to the current disclosure is the traditional notion of usingthe virus (considered a pathogen in this context) for study anddevelopment of genetic strategies against viruses. But discussed atfurther length in the present disclosure is the use of purified virusesas antigen carriers to prepare a vaccine. Antigens are molecules that,when appropriately delivered to an organism, are capable of producing animmune response in that organism, by stimulating the production ofantibodies through binding with an antibody within the organism thatmatches the molecular structure of the antigen. Recombinant antigens areproduced from recombinant DNA, which through known techniques is clonedinto vectors which are then introduced into specific host cells, such asbacteria, mammalian cells, yeast cells, and plant cells, to name some.The recombinant antigen is then expressed using the host cell'stranslational apparatus. After expression, the recombinant antigen canbe harvested and attached to a virus via covalent bonds, through aprocess known as conjugation. Following conjugation of the antigen tothe virus, the virus can serve as a carrier to deliver the antigen to anorganism and activate the immune system response. In this way, avirus-antigen conjugate can provide a therapeutic use. Propervirus-antigen conjugation is needed for the antigen to activate animmune response that produces antibodies in the host cells of a sourceorganism. Purification of both the virus and antigen fosters this properconjugation.

Current methods to purify viruses generally are limited for use in smallbiochemical quantities, e.g., on the order of nanograms to milligrams,and have not been proven in industrial quantities, which are on theorder of grams to kilograms. For example, a previously used method knownas “Crude Infected Cell Lysate” utilizes crude cell lysates or cellculture media from virus-infected cells. Infected mammalian cells arelysed by freeze-thaw or through other known methods, the debris isremoved by low-speed centrifugation, and supernatants are then used forexperimentation. The intact infected organisms are ruptured or groundphysically, and the resulting extract is clarified using centrifugationor filtration to produce crude virus preparations. However, this methodsuffers from high contamination with many non-virus factors that impactthe ability to conduct experimentation and manipulate the virus.

A second example of prior purification steps is high-speedultracentrifugation, by which viruses are pelleted, or further purifiedthrough pelleting, via a low-density sucrose solution, or suspended inbetween sucrose solutions of various densities. Limitations of thismethod include production of purified viruses in only small quantitiesdue to the limited size and scalability of high velocity separations,and poor virus purity due to additional host proteins often co-purifyingwith virus samples.

A third method previously used to enhance virus purity is densitygradient ultracentrifugation. In this method, gradients of cesiumchloride, sucrose, iodixanol or other solutions are used for separationof assembled virus particles or for removal of particles lacking geneticcontent. Limitations of this method include the time required to purifythe virus (often 2-3 days), the limited number of samples, the amount ofsamples that can be analyzed at a time (generally 6 per rotor), and thesmall quantity of virus that can be purified (generally micrograms tomilligrams of final product).

Organic extraction and poly-ethylene glycol precipitation also have beenused to purify viruses, including viruses from plants, such as byremoving lipids and chloroplasts. Again, however, these known methodssuffer from poor purity, with products typically still attached to hostproteins, nucleic acids, lipids, and sugars which result in significantaggregation of resulting virus products. These limitations reduce theutility of the final product for compliance with the Current GoodManufacturing Practice (cGMP) regulations enforced by the US Food andDrug Administration (FDA).

Current cGMP regulations promulgated by FDA contain minimum requirementsfor the methods, facilities, and controls used in manufacturing,processing, and packing of a drug product. These regulations are aimedat safety of a product and ensuring that it has the ingredients andstrength it claims to have. Accordingly, for viruses to be utilized invaccine creation, gene therapy, cancer treatments, and other clinicalsettings, the final viral product must comply with the cGMP regulations.If a final viral product does not comply with the cGMP regulations, likethe product from the poly-ethylene glycol precipitation method, itsutility for use in the clinical setting either does not exist or isgreatly diminished.

Scalability refers to a process that consistently and reproduciblyproduces the same product even as the quantity of product increases,e.g., going from laboratory scale (<0.1 square meters) to at leastsystems>20 square meters. The methods previously used as identifiedabove all suffer from a lack of consistency, low scalability (i.e.,creates product only in biochemical quantities), and a lack ofcompliance with the cGMP regulations.

In terms of large-scale production, plant-based production has garneredattention, although prominent limitations exist with their use.Plant-based production systems are capable of producing industrial scaleyields at much less cost than animal cell production systems such asChinese Hamster Ovary (CHO). However, certain conventional purificationmethods, which have been appropriate at some scale for non-plantviruses, will not work for plant-made viruses and antigens. Theselimitations arise because of myriad differences in purifying plantviruses, as opposed to the purification of viruses from animal cellcultures. While animal cells produce primary protein and nucleic acidimpurities, plants are also sources of significant and additionalimpurities not found in animal cells. Some of these include lipidcomposition of chloroplast membranes and vacuolar membranes, simple andcomplex carbohydrate impurities, and nano-particulate organellarimpurities. Indeed, crude plant extracts will often foul the equipmentused in processing and purifying the viral and antigen matter obtainedfrom plants, for example due to accumulation of impurities on theseparation membranes of the equipment or media beds leading. Suchfouling inevitably leads to pressure flow failure, poor filtration andultimately poor yield of product. Another problem is these impuritieshave a tendency to aggregate and become capable of co-purifying withinany protein, virus, or other “product” desired from a plant.Accordingly, current methods for purifying viruses will not adequatelyremove all or even a sufficient amount of impurities, including but notlimited to impurities found in plant extracts and have not been shown toadequately produce purified viruses.

Few advances have been seen for virus and antigen purification platformsconsistently capable of producing highly purified viruses on thecommercial scale, i.e. grams to kilograms and higher, and in a mannerthat complies with the cGMP regulations. Such improvements would allowfor the clinical development for using tools in vaccine creation, genetherapy, and for cancer treatments. Along with other features andadvantages outlined herein, the platforms described herein according tomultiple embodiments and alternatives meet this and other needs.

To date, seven coronaviruses (CoVs) have been identified as beingcapable of human infection, including severe acute respiratory syndromecoronavirus (SARS-CoV), Middle East respiratory syndrome coronavirus(MERS-CoV), and the newly identified CoV (SARS-CoV2, 2019-nCoV, orCovid-19), When in the human population, these three viruses posesignificant public health risks and exhibit high fatality rates:SARS-CoV: 10%, MERS-CoV: 34.4% and SARS-CoV2: 6.1%. Currently, Covid-19has spread to more than 188 countries, infected more than 10 millionpeople, and has been attributed to more than 500,000 deaths worldwide.These numbers are growing exponentially and the outbreak has created aworld-wide health and economic crisis. The emergence of Covid-19, andits effects on human health and the economy, demand an urgent response,particularly a vaccine for prevention of Covid-19 Disease. In spite of aglobal effort to find a vaccine to control or slow infections, noantiviral therapeutics are currently approved to target humancoronavirus. Instead, the primary treatment remains supportive andpalliative care.

The development of effective SARS-CoV and MERS-CoV vaccines has alsobeen met with limited success. Many traditional vaccine strategies havebeen utilized including inactivated virus, recombinant attenuatedviruses, other live viral vectors, subunit vaccines or individual viralproteins expressed from DNA plasmids or RNA delivery systems.Traditional SARS vaccines have primarily focused on the Spike (5)protein due to its functionality in human receptor binding, membranefusion, and viral entry. Furthermore, the S protein is the major antigenof coronaviruses and is the binding site of protective neutralizingantibodies that block virus receptor binding and initiation ofinfection. Although inactivated SARS-CoV preparations utilizingfull-length S proteins induced neutralizing antibodies in immunizedanimals, these conventional vaccines were not as effective in humans andhave been found to raise significant safety concerns (e.g. by actuallyenhancing viral infection in many systems). Likewise, while manydifferent SARS and MFRS vaccines have been developed and tested, allhave shown protection from infection through virus neutralizationwithout accompanying immunopathology associated with full-length ortrimerized S protein vaccines.

It will be appreciated that different viral vectors are known ascarriers for a range of antigens, providing effective therapeuticdelivery to a host organism, e.g., a mammal, such as a human. Such viralvectors tend to vary, however, in terms of an immune responses elicitedby the vector separate from the particular antigen being delivered. Forexample, some viral vectors, such as the ERVEBO® Ebola Zaire Vaccine,are live viruses, which stimulate a response by the host's immunesystem. The response from the viral vector itself, however, in manycases will blunt the intended immune response to an antigen delivered bythe viral vector, for example by showing immune dominance of the oneantigen thereby preventing a response during later administration of thesame or similar vaccine. Advantageously, it has been found that antigensof the present disclosure can be conjugated with a TMV NtK vectorwithout the latter stimulating the host's immune response, withoutshowing immune dominance of one antigen, and without affecting laterdose administration for subsequent vaccines using the same viral vector.Also, avoiding such an immune response, caused by the viral vectoritself, provides further advantages for bivalent, trivalent, andquadrivalent vaccines because the response for each antigen can beassessed without having to account for effects of the viral vector, bothin the current administration of the vaccine as well as futureadministrations.

Accordingly, there is a significant, urgent, and global need for aneffective and scalable vaccine strategy for the prevention of Covid-19Disease which both elicits high levels of neutralizing antibodies andinduces long lasting, potent neutralizing antibody titers for longperiods after immunization. Along with other features and advantagesoutlined herein, the platforms described herein according to multipleembodiments and alternatives meet this and other needs. In doing so, avaccine in accordance with present embodiments has exhibited strongimmune responses in pre-clinical studies, has the ability to conjugateto multiple CoV antigens at once (i.e. a multivalent vaccine) which is asignificant advantage over conventional monovalent vaccines, elicitshigh levels of neutralizing antibodies, and likely induces long lasting,potent neutralizing antibody titers for long periods after immunization.

SUMMARY OF EMBODIMENTS

In some embodiments according to the present disclosure, a viruspurification method is directed to a multi-set process that comprisesharvesting from a source organism virus material containing at least onevirus; removing cellular debris from the at least one virus therebyclarifying the structure of the at least one virus; concentrating theseparated and clarified virus which in some embodiments is performedwith a filtration device comprising a membrane with pores of a size notto exceed a predetermined limit as selected by a user; and processingthe concentrated virus by subjecting it to a series of separationprocedures and collecting the virus after each separation procedure,wherein at least one separation procedure includes ion-exchangechromatography to separate host cell contaminants from the virus, and atleast one separation procedure includes a multi-modal chromatography toseparate residual impurities from the virus on the basis of at leastsize differences between the virus and the impurities, and chemicalinteraction occurring between the impurities and one or morechromatography ligands. In some embodiments, a plant is the sourceorganism undergoing recombinant expression of a virus, with Nicotianabenthamiana and Leinna minor as non-limiting examples. When the sourceorganism is a plant, harvesting may include seed production and plantgermination with inducement of transient gene expression to from adesired protein, as discussed below. Alternatively, the source organismundergoing recombinant expression of a virus is a non-plant host suchas, without limitation, bacterial, alga yeast, insect, or mammalianorganisms.

Additionally, various aspects of multiple embodiments described hereinare directed to producing or purifying, or both, an antigen which can beconjugated with a virus particle. In the present embodiments andalternatives, a virus particle includes without limitation, one of, someof, or all of viruses and/or fragments thereof, such as rod-shapedviruses, icosahedral viruses, enveloped viruses, and fragments of one ormore of the foregoing. In some embodiments, a plant is the sourceorganism undergoing recombinant expression of antigen; alternatively,the source organism undergoing recombinant expression of antigen is anon-plant host such as, without limitation, bacterial, algal, yeast,insect, or mammalian organisms.

Advantageously, a multi-set process practiced according to variousembodiments described herein produces highly purified viruses orrecombinant antigens, or both, on a commercial scale. Various steps areemployed to improve the upstream purification processes, such asenriching plant viruses. Some embodiments utilize size exclusionchromatography, as well as other features, to produce purifiedrecombinant viruses and recombinant antigens. Accordingly, variousembodiments described herein provide one or more viruses and one or moreantigens suitable for the preparation of one or more vaccines ofconjugated virus and antigen.

With regard to viruses, through the practice of some embodiments of aninventive virus purification platform described herein, purification ofrod-shaped plant viruses (such as tobacco mosaic virus, i.e., “TMV”) andicosahedral plant viruses (such as red clover mosaic virus) has beenachieved. According to multiple embodiments herein, purification of TMVand red clover mosaic virus was achieved, representing two structurallydiverse viruses in terms of size and structure. For example, a smallericosahedral virus like red clover mosaic virus has T=3 symmetry,dimensions of approximately 31-34 nm, and approximately 180 capsidproteins. Conversely, TMV is approximately 18 nm in diameter, 300 nm inlength and contains 2160 capsid proteins. In view of this diversity, theinventive process has worked based on two structurally different virusesto allow virus passage into the permeate while retaining unwantedcellular debris. In use, operational parameters can be controlled so alltypes of viruses both pass into the permeate, while chlorophyll/cellulardebris are retained, and the tangential flow (TFF) system continues tooperate efficiently without unduly or untimely becoming fouled.Additional TFF steps are designed to retain virus while allowing smallerproteins to pass into the permeate, and dual chromatography steps arecontrolled to exclude viruses both large and small, while capturing hostcell proteins, host cell DNA, endotoxin, and plant polyphenolics.

Based upon the successful purification of red clover mosaic virus andTMV, it is expected that the virus purification platform according tomultiple embodiments and alternatives can successfully purify a widearray of virus particles including: viruses comprising a range ofgenetic materials (e.g. double- and single-stranded DNA viruses, and RNAviruses), geometries (e.g. rod-shaped, flexious rods, and icosahedral),and families (Caulimoviridae, Geminiviridae, Bromoviridae,Closteroviridae, Comoviridae, Potyviridae, Sequiviridae, Tombusviridae).

Non-limiting viruses upon which the embodiments described herein areexpected to succeed include those of the genuses Badnavirus (e.g.commelina yellow mottle virus); Caulimovirus (e.g. cauliflower mosaicvirus); SbCMV-like viruses (e.g. Soybean chlorotic mottle virus);CsVMV-like viruses (e.g. Cassava vein mosaicvirus); RTBV-like virusesrice tungro bacilliformvirus); petunia vein clearing-like viruses (e.g.petunia vein clearing virus); Mastrevirus (Subgroup I Geminivirus) (e.g.maize streak virus) and Curtovirus (Subgroup II Geminivirus) (e.g. beetcurly top virus) and Begomovirus (Subgroup III Geminivirus) (e.g. beangolden mosaic virus); Alfamovirus (e.g. alfalfa mosaic virus); Ilarvirus(e.g. tobacco streak virus); Bromovirus (e.g. brome mosaic virus);Cucumovirus (e.g. cucumber mosaic virus); Closterovirus (e.g. beetyellows virus); Crinivirus (e.g. Lettuce infectious yellows virus);Comovirus (e.g. cowpea mosaic virus); Fabavirus (e.g. broad bean wiltvirus 1); Nepovirus (e.g. tobacco ringspot virus); Potyvirus (e.g.potato virus Y); Rymovirus (e.g. ryegrass mosaic virus); Bymovirus (e.g.barley yellow mosaic virus); Sequivirus (e.g. parsnip yellow fleckvirus); Waikavirus (e.g. rice tungro spherical virus); Carmovirus (e.g.carnation mottle virus); Dianthovirus (e.g. carnation ringspot virus);Machlomovirus (e.g. maize chlorotic mottle virus); Necrovirus (e.g.tobacco necrosis virus); Tombusvirus (e.g. tomato bushy stunt virus);Capillovirus (e.g. apple stem grooving virus); Carlavirus (e.g.carnation latent virus); Enamovirus (e.g. pea enation mosaic virus);Furovirus (e.g. soil-borne wheat mosaic virus); Hordeivirus (e.g. barleystripe mosaic virus); Idaeovirus (e.g. raspberry bushy dwarf virus);Luteovirus (e.g. barley yellow dwarf virus); Marafivirus (e.g. maizerayado fino virus); Potexvirus (e.g. potato virus X and clover mosaicviruses); Sobemovirus Southern bean mosaic virus); Tenuivirus (e.g. ricestripe virus); Tobamovirus (e.g. tobacco mosaic virus); Tobravirus (e.g.tobacco rattle virus); Trichovirus (e.g. apple chlorotic leaf spotvirus); Tymovirus (e.g. turnip yellow mosaic virus; and Umbravirus (e.g.carrot mottle virus).

The successful virus purification has been accomplished on thecommercial scale, and in a manner that complies with the cGMPregulations. In some embodiments, the source organism is a plant, butwhile some variations of present embodiments include production ofplant-based viruses, the embodiments described herein are not limited tothe manufacture or the purification of viruses in plants. In someembodiments, a virus purification platform begins by growing plants in acontrolled growth room, infecting the plants with virus replication,recovering the viruses by rupturing the cells with a disintegrator andremoving the plant fiber from the liquid via a screw press.

In some embodiments, involving both plant-based and non-plant viruses,purification steps include concentrating the clarified extract usingtangential flow system, wherein the cassette pore size, transmembranepressure, and load of clarified extract per square meter of membranesurface area are controlled. Transmembrane pressure (IMP) is thepressure differential between the upstream and downstream sides of theseparation membrane and is calculated based on the following formula;((feed pressure+retentate pressure)/2)−permeate pressure. To ensurepassage of the viruses through the ceramic to create a clarifiedextract, in some embodiments the feed pressure, the retentate pressure,and the permeate pressure are each controlled to obtain an appropriateTMP. The clarified extract is concentrated further with an ion-exchangecolumn volume and washed with ion-exchange chromatography equilibrationbuffer. In some embodiments, a Capto Q ion-exchange column isequilibrated and the feed is loaded and collected in the flow-throughfraction. The column is then washed to baseline and the host cellcontaminants are stripped from the column with high salt.

In some embodiments associated with plant-based viruses, an extractionbuffer is added before removing chlorophyll and other large cellulardebris such as macro-molecular fibers, organelles, lipids, etc. usingtangential flow ceramic filtration. In some embodiments, ceramicfiltration promotes the retention of chlorophyll from plant hosts, celldebris, and other impurities while optimizing for virus passage. Whetherfor plant-based or non-plant viruses, this approach—wherein thedesirable matter (virus or antigen) passes through as permeate andimpurities are retained as retentate—promotes the scalability of theprocess. Additionally, parameters such as transmembrane pressure,ceramic pore size, and biomass loaded per square meter are allcontrolled to ensure passage of the virus through the ceramic to createa clarified extract. Ceramic TFF systems are highly scalable andparameters such as TMP, cross flow velocity, pore size, and surface areacan be scaled readily to accept larger amounts of biomass. Additionalceramic modules are easily added to the system. Feed, retentate, andpermeate pressure can also be controlled to maintain efficient crossflow velocity allowing little to no fouling of system. In someembodiments, cross velocity and pressure differential are set andcontrolled to produce a TMP of approximately 10-20 psi allowing forefficient passage of virus at smaller and larger scales. Ceramic TFFsystems are amenable to using highly efficient cleaning chemicals suchas nitric acid, bleach, and sodium hydroxide allowing for cleaningstudies to be performed addressing GMP and/or cGMP requirements.

Whether for plant-based or non-plant viruses, a purification methodaccording to multiple embodiments and alternatives, and otherwiseconsistent with the development of scalable and high-throughput methodsfor purifying viruses, utilizes at least one separation procedure usingmulti-modal chromatography to separate residual impurities from a viruson the basis of at least size differences between the virus and theimpurities, and chemical interaction occurring between the impuritiesand one or more chromatography ligands. For example, conducting the atleast one separation procedure with Capto® Core 700 chromatography resin(GE Healthcare Bio-Sciences) is included within the scope ofembodiments. The Capto® Core 700 ‘beads’ comprises octylamine ligandsdesigned to have both hydrophobic and positively charged properties thattrap molecules under a certain size, e.g. 700 kilodaltons (kDA). Becausecertain viruses are fairly large (e.g. greater than 700 kDA), and thebead exteriors are inactive, Capto® Core 700 permits purification ofviruses by size exclusion, wherein the desirable matter (virus orantigen) passes through as permeate and impurities are retained asretentate.

In some embodiments, again for plant-based and non-plant viruses alike,prior to the multi-modal chromatography column, equilibration isperformed with five column volumes of equilibration buffer. In someembodiments, the combined flow-through and wash fractions from Capto Qion-exchange chromatography are loaded onto the multi-modalchromatography column and the virus is collected in the void volume ofthe column. The column is washed to baseline and stripped with highconductivity sodium hydroxide. Aspects of some embodiments provide forcontrolling the loading ratio, column bed height, residence time, andchromatography buffers during this step.

The purified virus is sterile filtered, for example with diafiltration,and stored.

With regard to antigens, through the practice of some embodiments of aninventive antigen purification platform described herein, therecombinant antigens H5 recombinant influenza hemagglutinin (rHA), H7rHA, domain III of West Nile virus (WNV rDIII), and lassa fever virusrecombinant protein 1/2 (LFV rGP1/2), H1N1 (Influenza A/Michigan), H1N1(Influenza A/Brisbane), H3N2 (Influenza A/Singapore), H3N2 (InfluenzaA/Kansas), B/Colorado, B/Phuket, RBD-Fc 121 (receptor binding domain(RBD, S1 domain) of the SARS-2 spike glycoprotein fused to a human IgG1Fc domain (herein “RBD-Fc 121” refers an amino acid sequence of theSARS-2 spike protein as explained in further detail below)), and RBD-Fc139 (herein “RBD-Fc 139” refers to a different amino acid sequence ofthe SARS-2 spike protein, as explained below) have been produced andpurified. Antigens for various embodiments herein can be from manysources, and may be produced using traditional recombinant proteinmanufacturing strategies, including bacterial, yeast, insect, mammalianor plant-based expression approaches.

In some embodiments, an antigen manufacturing platform begins by growingplants in a controlled growth room, infecting the plants for recombinantantigen replication, then antigen recovery using a disintegratorfollowed by removal of fiber from the aqueous liquid via a screw press.An extraction buffer is added to assist in removal of chlorophyll (inthe plant context) and large cellular debris by filtration. Whether forplant-based or non-plant antigen, feed pressure, filtrate pore size,clarifying agent, and biomass loaded per square meter of membranesurface are controlled to facilitate passage of the antigens through thefilter. A description (though non-limiting) of various in-processcontrols suitable for achieving large scale virus and antigenpurification is expressed in further detail in the Examples section.

In some embodiments, both plant-based and non-plant antigens alike,clarified extract is next concentrated with a tangential flow system.During this optional step, factors including cassette pore size,transmembrane pressure, and load of clarified extract per square meterof membrane surface are controlled. In some embodiments, the optionalstep is skipped entirely. Following this, clarified extract is nextconcentrated and washed with an ion-exchange chromatographyequilibration buffer. One way for this step to be undertaken is byloading feed onto an equilibrated Capto Q ion-exchange column, followedby washing with equilibration buffer and eluting/stripping with salt.Antigen fractions are then collected in the elution and prepared forcobalt immobilized metal affinity chromatography (IMAC). The IMAC isequilibrated, the feed is loaded, then washed with equilibration bufferand eluted. The elution fraction is diluted and checked for pH, thenloaded onto a multi-modal ceramic hydroxyapatite (CHT) chromatographycolumn. The CHT resin is equilibrated with equilibration buffer and theantigens are eluted. Loading ratio, column bed height, residence time,and chromatography buffers are among factors being controlled. Lastly,the antigen is concentrated and diafiltered with a saline buffer. Therecombinant antigen is sterile filtered and then stored.

Still further, in accordance with various embodiments disclosed herein,the following monovalent formulations have been successfully conjugated:H7 rHA to TMV, H1N1 (Influenza A/Michigan) to TMV, H3N2 (InfluenzaA/Singapore) to TMV, B/Colorado to TMV, B/Phuket to TMV, RBD-Fc 121(SARS-2) to TMV, and RBD-Fc 139 (SARS-2) to TMV. In accordance with thevarious embodiments herein, the bivalent formulation of TMV to twoInfluenza B viruses (B/Colorado and B/Phuket) has also been successfullyconjugated, as well as the quadrivalent conjugation of TMV to H1N1(Influenza A/Michigan), H3N2 (Influenza A/Singapore), B/Phuket, andB/Colorado. A “quadrivalent” influenza vaccine is designed to protectagainst four different influenza viruses: two influenza A viruses andtwo influenza B viruses. For many years, trivalent vaccines werecommonly used, but now quadrivalent vaccines are the most common becausethey may beneficially provide broader protection against circulatinginfluenza viruses by adding another B virus. Herein, the term“multivalent” vaccine refers to more than one antigen conjugated to avirus. In some embodiments, the protein consists of any type oftherapeutic agent capable of being conjugated to a virus to create avaccine, and then delivered to a source organism to produce an immuneresponse according to multiple embodiments and alternatives.Accordingly, the disclosures herein provide compositions comprising anarray of virus-protein conjugates, including virus-antigen conjugates.In some embodiments, the virus selected is TMV, or any of a number ofviruses identified and/or indicated by the teachings herein.Additionally, in some embodiments the protein can be an antigen, such asbut not limited to influenza hemagglutinin antigen (HA), includingwithout limitation ones listed in this paragraph including as solubleforms of HA proteins found on a surface of an influenza virus thatmediates virus infection. In some embodiments, the HA exhibits at leastabout 50% trimer formation. HAs are clinically important because theytend to be recognized by certain antibodies an organism produces,providing the main thrust of protection against various influenzainfections. Because HA antigenicity and, therefore, HA immunogenicityare tied to conformation, it is known that HA trimerization isadvantageous over the monomeric form in terms of triggering immuneresponses.

In some embodiments, conjugation begins by concentrating anddiafiltering purified antigen and virus into a slightly acidic buffer.The antigen and virus are then combined based upon molarity and mixed. Afreshly prepared water-soluble carbodiimide, such as1-ethyl-3-(3-dimethylaminopropyl) carbodiimide (also known as EDC) isadded to the mixture while mixing based upon molarity. A chemicalreagent for converting carboxyl groups to amine reactiveN-hydroxysulfosuccinimide esters, such as ThermoFisher's Sulfo-NHS, isthen added based upon molarity. The reaction is continued until apredetermining stop time. The reaction is then quenched, with oneexemplary involving the addition of an amine group (e.g., liquidcontaining free amines) and any chemical linker(s) used in facilitatingthe reaction (e.g., EDC, Sulfo-NHS) is removed through a multi-modalchromatography step or diafiltration, with the mixture then beingdiluted to target concentration. In some embodiments, the conjugated andpurified virus particles that are decorated with proteins and antigensmay be used for vaccines and/or diagnostic tools. These particles may beused as diagnostic tools because of their ability to track antigens inthe host organism.

In some embodiments, the purified virus—antigen fusion may be derivedfrom genetic fusion, in addition to the various embodiments disclosedherein. The antigen and virus structural proteins (located in the coat)form a single continuous open reading frame. In some embodiments, thereading frame produces an antigen-coat protein in a plant such that thecoat protein self assembles into virus particles. Next, the plantmaterials are harvested and the virus particles are purified accordingto the embodiments disclosed herein. The virus particles decorated withthe fusion-coat proteins may then be used as a vaccine and/or adiagnostic tool according to the various embodiments disclosed.

Some viruses as icosahedral viruses as a non-limiting example) swellunder certain pH conditions and in some embodiments this “swelling” maybe used for conjugation. According to multiple embodiments andalternatives, the purified virus may be conjugated to a therapeuticagent by subjecting the virus structure to acidic pH conditions thatcause the virus to “swell.” By treating the virus structure with neutralpH conditions, the virus structure relaxes and creates pores betweenpentamer or other structural subunits of the virus. Next, a therapeuticagent (such as a chemotherapeutic agent), is added to the buffer andallowed to diffuse into the relaxed virus particle. By changing the pHagain, the virus particles tighten and remove the pore structurespacking the pentamer or structural submits together such that chemicaldiffusion in or out of the virus particle is prevented. Next, the plantmaterials are harvested, the virus particles are purified, and the virusparticles containing a therapeutic agent are used for drug delivery,according to the embodiments disclosed herein.

Accordingly, multiple embodiments and alternatives encompass productionof one or more highly purified viruses. Still further, multipleembodiments and alternatives encompass production or purification orboth of a recombinant antigen. Still further, multiple embodiments andalternatives encompass conjugation of purified antigens and viruses foruse as vaccines. Indeed, in pre-clinical studies, TMV-platform vaccinesproduced in accordance with the embodiments described herein stimulatedefficacious immune responses against a number of pathogens, comprisingboth viral and antibacterial systems. Further, vaccines produced on theinventive TMV-platform demonstrate the ability to conjugate to multiplecoronavirus (CoV) antigens at once (i.e. a multivalent vaccine) tostimulate efficacious immune responses. This provides a significantadvantage over conventional monovalent vaccines, including through theapplication of these vaccines against Covid-19 Disease and influenza (asnon-limiting examples). The purification of viruses may be practiced byitself in accordance with the present embodiments. Likewise, theproduction or purification of recombinant antigens may be practicedalone in accordance with the present embodiments. Optionally, as well,different aspects of these multiple embodiments can be combined, inwhich combining embodiments would include, among other ways ofpracticing these embodiments, starting with one or more sourceorganisms, from which are produced one or more viruses and one or moreantigens, then purifying such viruses and antigens, then formingvaccines which are conjugates between at least one antigen and at leastone virus.

BRIEF DESCRIPTION OF THE FIGURES

The drawings and embodiments described herein are illustrative ofmultiple alternative structures, aspects, and features of the multipleembodiments and alternatives disclosed herein, and they are not to beunderstood as limiting the scope of any of these embodiments andalternatives. It will be further understood that the drawing figuresdescribed and provided herein are not to scale, and that the embodimentsare not limited to the precise arrangements, depictions, andinstrumentalities shown.

FIG. 1 is a flow chart showing the steps in a certain virus purificationplatform within the scope of the present disclosure, according tomultiple embodiments and alternatives.

FIG. 2 is purified icosahedral red clover mosaic virus, according tomultiple embodiments and alternatives.

FIG. 3 is a western blot analysis of the purification of the icosahedralred clover mosaic virus, according to multiple embodiments andalternatives.

FIG. 4 is purified icosahedral red clover mosaic virus, according tomultiple embodiments and alternatives.

FIG. 5 is a western blot analysis of the purification of the icosahedralred clover mosaic virus, according to multiple embodiments andalternatives.

FIG. 6 is purified rod-shaped tobacco mosaic virus, according tomultiple embodiments and alternatives.

FIG. 7 is a western blot analysis of the purification of the rod-shapedtobacco mosaic virus, according to multiple embodiments andalternatives.

FIG. 8 is a flow chart showing the steps of an antigen manufacturingplatform, according to multiple embodiments and alternatives.

FIG. 9 is a western blot analysis of some of the steps of an antigenmanufacturing platform, according to multiple embodiments andalternatives.

FIG. 10 is a western blot analysis of some of the steps of an antigenmanufacturing platform, according to multiple embodiments andalternatives.

FIG. 11 is a western blot analysis of some of the steps of an antigenmanufacturing platform, according to multiple embodiments andalternatives.

FIG. 12 is a western blot analysis of the purification of variousantigens through the antigen manufacturing platform, according tomultiple embodiments and alternatives.

FIG. 13 is an illustration of the conjugation of recombinant antigen toa virus, according to multiple embodiments and alternatives.

FIG. 14 is a SDS-PAGE analysis of the conjugation of an antigen to avirus, according to multiple embodiments and alternatives.

FIG. 15 is a SDS-PAGE analysis of the conjugation of an antigen to avirus, according to multiple embodiments and alternatives.

FIG. 16 is a SDS-PAGE analysis of the conjugation of an antigen to avirus, according to multiple embodiments and alternatives.

FIG. 17 is a report of size exclusion-high-performance liquidchromatography (SEC-HPLC) of a free TMV product, according to multipleembodiments and alternatives.

FIG. 18 is a report of SEC-HPLC of conjugation between a virus and anantigen for fifteen minutes, according to multiple embodiments andalternatives.

FIG. 19 is a report of SEC-HPLC of conjugation between a virus and anantigen two hours, according to multiple embodiments and alternatives.

FIG. 20 is a western blot analysis of conjugation between a virus and anantigen, according to multiple embodiments and alternatives.

FIG. 21 is a graph illustrating the infectivity of viruses treated withvarious levels of UV irradiation, according to multiple embodiments andalternatives.

FIG. 22 is an illustration of some of the steps of the conjugationplatform of recombinant antigen to a virus, according to multipleembodiments and alternatives.

FIG. 23 is a SDS-PAGE analysis of the conjugation of an antigen to avirus, according to multiple embodiments and alternatives.

FIG. 24 is a negative stain transmission electron microscopy (TEM) imageof recombinant antigen, according to multiple embodiments andalternatives.

FIG. 25 is a negative stain TEM image of a virus, according to multipleembodiments and alternatives.

FIG. 26 is a negative stain TEM image of a recombinant antigenconjugated to another recombinant antigen with added virus, according tomultiple embodiments and alternatives.

FIG. 27 is a negative stain TEM image of recombinant antigen conjugatedto a virus at a virus to recombinant antigen ratio of 1:1, according tomultiple embodiments and alternatives.

FIG. 28 is a negative stain TEM image of recombinant antigen conjugatedto a virus at a virus to recombinant antigen ratio of 1:1, according tomultiple embodiments and alternatives.

FIG. 29 is a negative stain TEM image of recombinant antigen conjugatedto a virus at a virus to recombinant antigen ratio of 4:1, according tomultiple embodiments and alternatives.

FIG. 30 is a negative stain TEM image of recombinant antigen conjugatedto a virus at a virus to recombinant antigen ratio of 16:1, according tomultiple embodiments and alternatives.

FIG. 31 is a normalized sedimentation coefficient distribution of anantigen, according to multiple embodiments and alternatives.

FIG. 32 is a normalized sedimentation coefficient distribution of avirus, according to multiple embodiments and alternatives.

FIG. 33 is a normalized sedimentation coefficient distribution ofrecombinant antigen conjugated to a virus at a virus to recombinantantigen ratio of 1:1, according to multiple embodiments andalternatives.

FIG. 34 is a normalized sedimentation coefficient distribution ofrecombinant antigen conjugated to a virus at a virus to recombinantantigen ratio of 1:1, according to multiple embodiments andalternatives.

FIG. 35 is a normalized sedimentation coefficient distribution ofrecombinant antigen conjugated to a virus at a virus to recombinantantigen ratio of 1:1, according to multiple embodiments andalternatives.

FIG. 36 is a normalized sedimentation coefficient distribution ofrecombinant antigen conjugated to a virus at a virus to recombinantantigen ratio of 4:1, according to multiple embodiments andalternatives.

FIG. 37 is a normalized sedimentation coefficient distribution ofrecombinant antigen conjugated to a virus at a virus to recombinantantigen ratio of 16:1, according to multiple embodiments andalternatives.

FIG. 38 is a scatterplot of antigen-relevant titers in a source organismfollowing administration of virus-antigen products at various virus torecombinant ratios, according to multiple embodiments and alternatives.

FIG. 39 is a geometric mean testing illustrating the antigen-relevanttiters in a source organism following administration of virus-antigenproducts at various virus to recombinant ratios, according to multipleembodiments and alternatives.

FIG. 40 is a SDS-PAGE analysis of a purified recombinant antigen,according to multiple embodiments and alternatives.

FIG. 41 is a graph of the immunogenicity of a quadrivalent vaccine inmice, according to multiple embodiments and alternatives.

FIG. 42 is an illustration of an immunogenicity and challenge study of aquadrivalent vaccine in ferrets, according to multiple embodiments andalternatives.

FIG. 43 is an illustration of the nasal wash virus titers followingvirus challenge in ferrets, according to multiple embodiments andalternatives.

FIG. 44 is an illustration of the nasal wash virus titers followingvirus challenge in ferrets, according to multiple embodiments andalternatives.

FIG. 45 is an illustration of the immunogenicity of a monovalent vaccineproduced in mice at various virus to recombinant antigen ratios,according to multiple embodiments and alternatives.

FIG. 46 is an illustration of TMV vRNA in tissues over time following aninjection of a quadrivalent vaccine, according to multiple embodimentsand alternatives.

FIG. 47 is an illustration of TMV vRNA in tissues eight days followingan injection of a quadrivalent vaccine, according to multipleembodiments and alternatives.

FIG. 48 is an illustration of total anti-influenza titers based on anELISA analysis from rabbit serum samples following injections of aquadrivalent vaccine, according to multiple embodiments andalternatives.

FIG. 49 is an illustration of neutralization titers measured in rabbitsfollowing injections of a quadrivalent vaccine, according to multipleembodiments and alternatives.

FIG. 50 (A) is an illustration of the protein structure of the Covid-19Spike timer in space-filling model with the receptor binding domaincircled in lateral and vertical views. FIG. 50(B) is an illustration ofthe fusion of the Covid-19 receptor binding domain to a human Fc domain,according to multiple embodiments and alternatives.

FIG. 51 is an illustration of an expression plasmid containing theconstruct of a recombinant Covid-19 antigen, according to multipleembodiments and alternatives.

FIG. 52 is a SDS-PAGE analysis of a purified recombinant Covid-19antigen, according to multiple embodiments and alternatives.

FIG. 53 is a report of SEC-HPLC of a purified recombinant Covid-19antigen, according to multiple embodiments and alternatives.

FIG. 54 is a SUS-PAGE analysis of a purified recombinant Covid-19antigen, according to multiple embodiments and alternatives.

FIG. 55 is an illustration of some of the steps of the conjugationplatform of recombinant Covid-19 antigen to a virus and drug substancefilling, according to multiple embodiments and alternatives.

FIG. 56 is a normalized sedimentation coefficient distribution of arecombinant Covid-19 antigen conjugated to a virus, according tomultiple embodiments and alternatives.

FIG. 57 is a normalized sedimentation coefficient distribution of arecombinant Covid-19 antigen conjugated to a virus, according tomultiple embodiments and alternatives.

FIG. 58 is a SDS-PAGE analysis of a recombinant Covid-19 antigenconjugated to a virus, according to multiple embodiments andalternatives.

FIG. 59(A) is an ELISA standard curve illustrating the binding of therecombinant Covid-19 antigen to a Covid-19 human neutralizing monoclonalantibody. FIG. 59(B) is a graph illustrating the binding of variousvaccine alternatives in accordance with multiple embodiments andalternatives described herein (sometimes referred to herein as vaccinecandidates), and the binding to a Covid-19 human neutralizing monoclonalantibody, according to multiple embodiments and alternatives.

FIG. 60 consists of confocal microscopy images illustrating theco-localization of the recombinant Covid-19 antigen to ACE-2 specificantibodies, according to multiple embodiments and alternatives.

FIG. 61(A) is a graph illustrating the co-localization of a nativeagonist and a recombinant Covid-19 antigen to ACE-2 specific antibodies,according to multiple embodiments and alternatives. FIG. 61(B) is agraph illustrating the co-localization of a native agonist and arecombinant Covid-19 antigen to ACE-2 specific antibodies in thepresence of a co-localization control, according to multiple embodimentsand alternatives.

FIG. 62(A) is a graph illustrating the immune response in animalsimmunized with a recombinant Covid-19 antigen and a control. FIG. 62(B)is a graph illustrating the immune response in animals immunized with arecombinant Covid-19 antigen conjugated to a virus (unadjuvanted). FIG.62(C) illustrates the immune response in animals immunized with anadjuvanted Covid-19 antigen conjugated to a virus. FIG. 62(D) comparesthe IgG titers recognizing the Covid-19 antigen (in black) and RBD-His(in gray).

FIGS. 63(A)-63(C) are graphs illustrating an IgG isotype analysis forindividual sera taken from animals immunized with a recombinant Covid-19antigen conjugated to a virus, controls, and adjuvant, according tomultiple embodiments and alternatives. FIG. 63(A) illustrates the Th1response (IgG2a and IgG2c) associated with the analysis, FIG. 63(B)illustrates the Th2 response (IgG1) associated with the analysis, andFIG. 63(C) is a stack plot comparison of relative IgG2 versus IgG1antibody response by an ELISA analysis.

FIG. 64 is a graph illustrating the cell viability after incubation withSARS2 virus with murine sera, according to multiple embodiments andalternatives.

FIG. 65 is a graph illustrating the immune response titers in animalsimmunized with a recombinant Covid-19 antigen conjugated to a virus,controls, and historical comparisons, according to multiple embodimentsand alternatives.

FIG. 66(A) is a graph illustrating an IFNγ ELISpot analysis for animalsimmunized with a virus and a recombinant Covid-19 antigen. FIG. 66(B) isa graph illustrating an IFNγ ELISpot analysis for animals immunized withan unadjuvanted TMV:RBD-Fc vaccine. FIG. 66(C) is a graph illustratingan IFNγ ELISpot analysis for animals immunized with a TMV:RBD-Fc vaccineand an adjuvant.

FIG. 67 is a graph illustrating a VaxArray® analysis of murine sera frommice immunized with various TMV:RBD-Fc vaccine dosages, and both withand without adjuvant, according to multiple embodiments andalternatives.

FIG. 68(A) is a graph showing results on the viability of macrophagesexposed to nucleocapsid and spike proteins of SARS-2 with non-inventiveantibodies added, as an indicator of antibody enhancement of disease.FIG. 68(B) is a graph of mouse sera containing antibodies, which weregenerated from TMV:RBD-Fc vaccines according to multiple embodiments andalternatives, following exposure to nucleocapsid and spike proteins ofSARS-CoV2. FIG. 68(C) shows statistical differences applicable to thegraphs in FIGS. 68 (A-B).

FIG. 69 is a SUS-PAGE analysis of a purified recombinant Covid-19antigen, according to multiple embodiments and alternatives.

FIG. 70 is a report of SEC-HPLC of a purified recombinant Covid-19antigen, according to multiple embodiments and alternatives.

FIG. 71 illustrates the receptor binding domain of the spike proteinsequence alignment of SARS-2 and other related coronaviruses.

MULTIPLE EMBODIMENTS AND ALTERNATIVES

A multi-set process according to multiple embodiments and alternativesherein improves upstream purification processes, further enriching plantviruses, and facilitates the conjugation of virus and antigen to form avaccine. Steps for producing and purifying a virus in accordance withmultiple embodiments and alternatives are listed and discussed inconnection with Table 1 and FIG. 1 . Likewise, steps for producing andpurifying an antigen are listed and discussed in connection with Table2. Although the various platforms have a specific embodiment describedfor them below, the scope of the embodiments contained herein are notlimited to any one specific embodiment.

Virus Production and Purification

Table 1 and FIG. 1 illustrate the steps of the virus purificationplatform according to multiple embodiments and alternatives.

TABLE 1 Production and Purification of Virus Operative Steps UnitOperations In-Process Controls In-Process Analytics  1 Plant Growth (25DPS) Irrigation, Light Plant Height, structure Nb Cycle, Fertilizer, andleaf quality Media, Humidity, Temperature  2 Infection with virusInoculum N/A Concentration, Rate of Application  3 Viral Replication (7Irrigation, Light N/A DPI) Plant Growth Cycle, Humidity, Temperature  4Harvest of Aerial Tissue Visual Inspection of N/A Plants  5Disintegration of Plant Blade Type and RPM, pH, Conductivity, Cells(Extraction) Screen Sizes, SDSPage, Endotoxin, Buffer:Tissue RatioNicotine  6 Clarification of Plant Ceramic Size, TMP, pH, Conductivity,Extract kg/m² SDSPage, Endotoxin, Nicotine  7 Concentration of PoreSize, TMP, Pore pH, Conductivity, Clarified Plant Extract Material,kg/m² SDSPage, Endotoxin, Nicotine  8 Ion-Exchange kg/L, Bed Height, pH,Conductivity, Chromatography Residence Time SDSPage, Endotoxin, Nicotine 9 Multi-Modal kg/L, Bed Height, pH, Conductivity, ChromatographyResidence Time SDSPage, Endotoxin, Nicotine 10 Concentration of PoreSize, TMP, Pore UV260, TEM, DLS, Purified Virus Material, kg/m² SDSPage,Endotoxin, Nicotine, Amino Acid

This purification platform is designed for commercial scalability andcompliance with the cGMP regulations and utilizes one buffer throughoutthe entire purification process. According to multiple embodiments andalternatives, the steps of the virus purification platform are given inconnection with plant expression. However, steps after the aerial tissueharvesting and cell rupture as described below also would apply tonon-plant viruses (except where context is clearly related to plants,e.g., reference to oval of plant fiber).

In accordance with multiple embodiments and alternatives describedherein, virus expression is accomplished through methods that areappropriate for a particular host. In some embodiments, virus-baseddelivery of genes to a plant host is accomplished with a modified TMVexpression vector that causes tobacco plants to recombinantly form thevirus. One such available alternative is the GENEWARE® platformdescribed in U.S. Pat. No. 7,939,318, “Flexible vaccine assembly andvaccine delivery platform.” This transient plant-based expressionplatform described in this patent employs the plant virus TMV to harnessplant protein production machinery, which expresses a variety of virusesin a short amount of harvest time post inoculation (e.g., less than 21days). Tobacco plants inoculated with the virus genes express theparticular virus in infected cells, and the viruses are extracted atharvest. Inoculation occurs by, as examples to be selected by a user ofthe methods herein described, hand inoculation of a surface of a leaf,mechanical inoculation of a plant bed, a high-pressure spray of a leaf,or vacuum infiltration.

Besides Nicotiana benthamiana, other plant and non-plant hosts arecontemplated by this disclosure, including those mentioned in theSummary. Besides the GENEWARE® platform, other strategies can beemployed to deliver genes to plant (Lemna gibba or Lemna minor asnon-limiting examples) and non-plant organisms (algae as a non-limitingexample). These other strategies include Agro-infiltration, whichintroduces the viral gene via an Agrobacterium bacterial vector to manycells throughout the transfected plant. Another is electroporation toopen pores in the cell membranes of the host to introduce the genes thatrecombinantly produce the viruses and antigens such as but not limitedto those described in Examples 1 and 3 below. Another is TMV RNA-basedoverexpression (TRBO) vector, which utilizes a 35S promotor-driven TMVreplicon that lacks the TMV coat protein gene sequence, as described inJohn Lingo, “TRBO: A High-Efficiency Tobacco Mosaic Virus RNA-BasedOverexpression Vector,” Plant Physiol. Vol. 145, 2007.

In some embodiments, growth of Nicotiana benthamiana wild type plantsoccurs in a controlled growth room. Plant growth is controlled viairrigation, light, and fertilized cycles. Plants are grown in a soillessmedia and temperature is controlled throughout the process.

After an appropriate number of days post sow (DPS), for example 23-25DPS, the plants are infected with the virus replication. Afterinfection, the plants are irrigated with water only and controlled vialight cycle and temperature for a certain number of days post infection(DPI) depending on the type of virus.

Plants are inspected for height, infection symptoms, and the aerialtissue is harvested.

Virus recovery/cell rupture involves a disintegrator configured with anoptimized blade/screen size followed by removal of residual cellulosicplant fiber from aqueous liquid (such as through a screw press, as oneexample).

An appropriate extraction buffer (e.g., 200 mM Sodium Acetate, pH 5.0;step 201 of FIG. 1 as a non-limiting example) is added to the resultingextract at a 1:1 buffer:tissue ratio. Removal of chlorophyll and largecellular debris at pilot scale involves the use of tangential flow (TFF)ceramic filtration (1.4 micron/5.0 micron). Transmembrane pressure,ceramic pore size and biomass loaded per square meter of membranesurface are all controlled to ensure passage of the virus through theceramic. In some embodiments, the feed pressure, retentate pressure, andpermeate pressure are set and controlled to produce a resultingtransmembrane pressure in a range of about 1.5-2 Bar TMP.

Ceramic permeate is further clarified via the use of glass fiber depthfiltration (step 203 of FIG. 1 as a non-limiting example).

Clarified extract is concentrated with a TFF system (available fromSartorius AG). Cassette pore size (100-300 kDa), an appropriate IMP asdescribed herein, and load of clarified extract per square meter ofmembrane surface area are controlled.

The clarified extract is concentrated to NMT 2× the ion-exchange columnvolume and washed 7× with ion-exchange chromatography equilibrationbuffer (200 mM Sodium Acetate, pH 5.0, step 204 of FIG. 1 provides anon-limiting example). The Capto Q ion-exchange column is equilibratedfor five column volumes with 200 mM Sodium Acetate, pH 5.0 (step 205 ofFIG. 1 provides a non-limiting example), and the feed is loaded andcollected in the flow-through fraction. The column is washed to baselineand host cell contaminants are stripped from the column with high salt.

The flow through and wash fractions are collected, combined and preparedfor multi-modal Capto® Core 700 chromatography. The multi-modalchromatography column is equilibrated with five column volumes ofequilibration buffer (200 mM Sodium Acetate, pH 5.0; step 206 of FIG. 1provides a non-limiting example).

The combined flow-through and wash fractions from Capto Q ion-exchangechromatography are loaded onto the column and the virus collected in thevoid volume of the column. The column is washed to baseline and strippedwith high conductivity sodium hydroxide. Loading ratio, column bedheight, residence time and chromatography buffers are all controlled.Formulation and concentration of virus (step 208, FIG. 2 ) takes placein some embodiments with a TFF System (such as the Sartorius AG system).Pore size (30-300 kDa), an appropriate TMP as described herein, load persquare meter of membrane surface area and pore material are allcontrolled. Virus is concentrated to an appropriate concentration, suchas 10 mg/ml, and in some embodiments is diafiltered with an appropriatebuffer, such as Sodium Phosphate. Formulated virus is sterilized andstored appropriately. In some embodiments, sterilization is provided viaa PES filter.

All examples provided herein are meant as illustrative of variousaspects of multiple embodiments and alternatives of any or all of virusproduction, virus purification, antigen production, antigenpurification, and virus-antigen conjugation. These examples arenon-limiting and merely characteristic of multiple alternativeembodiments herein.

Example 1—Purification of Icosahedral Red Clover Mosaic Virus

The Western Blot, provided in FIG. 3 as a known technique for detectingvarious proteins in a mixture, shows successful purification of theicosahedral red clover mosaic virus illustrated in FIG. 2 . Similarly,the Western Blot in FIG. 5 shows successful purification of theicosahedral red clover mosaic virus illustrated in FIG. 4 . Both viruseswere purified according to the embodiments described herein. Inaccordance with the known detection technique, target proteins wereextracted from the tissue. Then proteins of the sample were separatedusing gel electrophoreses based on their isoelectric point, molecularweight, electrical charge, or various combinations of these factors.Samples were then loaded into various lanes in the gel, with a lanereserved for a “ladder” containing a mixture of known proteins withdefined molecular weights. For example, in FIG. 3 , lane 12 serves asthe ladder. A voltage was then applied to the gel, causing the variousproteins to migrate through the gel at different speeds based on theaforementioned factors. The separation of the different proteins intovisible bands within each lane occurred as provided in FIGS. 3 and 5 ,respectively. With the Western Blot, a purer product is characterized bya clear and visible band, and such is characterized in these figures.

FIGS. 3 and 5 illustrate the virus purification platform successfullypurifying the icosahedral red clover mosaic virus. Each lane of thewestern blot shows the purity of the virus after the conclusion of adifferent step in the virus purification platform. In FIG. 3 , the lanesinclude: lane 1—green juice, lane 2—TFF Ceramic Clarification Retentate,lane 3—TFF Ceramic Clarification Permeate, lane 4 TFF CassetteRetentate, lane 5—TFF Cassette Permeate, lane 6—ion Exchange, lane 7—IonExchange, lane 8—multimodal, lane 9—multimodal, lane 10—30TFF Permeate,lane 11—30K Retentate, lane 12—marker. In FIG. 5 , the lanes of thewestern blot include the following: lane 1—Green Juice, lane 3—TFFCeramic Clarification Retentate, lane 5—TFF Ceramic ClarificationPermeate, lane 7—TFF Cassette Retentate, lane 9—TFF Cassette Permeate,lane 11—ion Exchange, lane 13—Multimodal, and Lane 14—Marker.

Once the final step has occurred in the virus purification platform, theresulting viral product is highly purified, as shown by the visible bandin lane 11 of FIG. 3 and lane 13 of FIG. 5 .

Example 2—Purification of Rod-Shaped TMV

FIG. 6 shows a purified rod-shaped TMV, and FIG. 7 illustrates a viruspurification platform used in achieving this purified TMV, within thescope of multiple embodiments and alternatives disclosed herein. Similarto FIGS. 3 and 5 , FIG. 7 illustrates the purity of the virus productafter the conclusion of the various steps of the current viruspurification platform. After the final purification step, the resultingproduct is highly purified virus product consistent with a clear andvisible band in lane 13 of FIG. 7 .

Accordingly, an inventive virus purification platform has successfullypurified every virus on which the inventors have applied these methods,including both an icosahedral virus and a rod-shaped virus, and thisplatform is expected to be reproducible and consistently purify on acommercial scale virtually any type (if not all types) of virus.

Production and Purification of Recombinant Antigen

Table 2 and FIG. 8 illustrate the steps of the antigen purificationplatform according to multiple embodiments and alternatives.

TABLE 2 Production and Purification of Recombinant Antigen OperativeIn-Process Steps Unit Operations In-Process Controls Analytics  1 PlantGrowth (25 UPS) Nb Irrigation, Light Cycle, Plant height, Fertilizer,Media, structure and Humidity, Temperature leaf quality  2 GENEWAREInfection Inoculum with Target Antigen Concentration, Rate ofApplication  3 Replication (7-14 DPI) Irrigation, Light Cycle, PlantGrowth Humidity, Temperature  4 Harvest of Aerial Tissue VisualInspection of Plants  5 Disintegration of Plant Blade Type and RPM, pH,Cells (Extraction) Screen Sizes, Conductivity, Buffer:Tissue RatioSDSPage, Endotoxin, Nicotine  6 Clarification of Plant Filter Press PoreSize, pH, Extract Feed Pressure, kg/m2 Conductivity, SDSPage, Endotoxin,Nicotine  7 Concentration of Clarified Pore Size, TMP, Pore pH, PlantExtract Material, kg/m² Conductivity, SDSPage, Endotoxin, Nicotine  8Capto Q Chromatography kg/L, Bed Height, pH, Residence TimeConductivity, SDSPage, Endotoxin, Nicotine  9 ColMAC or ConA kg/L, BedHeight, pH, Residence Time Conductivity, SDSPage, Endotoxin, Nicotine 10Ceramic Hydroxyapatite kg/L, Bed Height, pH, Residence TimeConductivity, SDSPage, Endotoxin, Nicotine 11 Concentration/FormulationPore Size, TMP, Pore UV260, TEM, of Purified Antigen Material, kg/m²DLS, SDSPage, Endotoxin, Nicotine, Amino Acid

This purification platform is designed for commercial scalability andcompliance with the cGMP regulations and utilizes one buffer throughoutthe entire purification process. According to multiple embodiments andalternatives, the steps of the antigen purification platform are asfollows:

Growth of Nicotiana benthamiana wild type plants in a controlled growthroom. Plant growth is controlled via irrigation, light and fertilizercycles. Plants are grown in a soilless media and temperature iscontrolled throughout the process. After an appropriate number of DPS,for example 23 to 25, plants are infected for protein replication of aselected antigen. Once tagged, the protein is sufficient for retentionin the ER of the transgenic plant cell. After infection plants areirrigated with water only and controlled via light cycle and temperaturefor an appropriate number of days post infection, such as 7-14 daysdepending on the type of antigen. Plants are inspected for height andinfection symptoms, and the aerial tissue is harvested.

Recovery of antigen produced by the plants involves a disintegratorconfigured with an optimized blade/screen size followed by removal ofresidual cellulosic plant fiber from aqueous liquid (such as through ascrew press, as one example).

A suitable extraction buffer is added to the resulting extract at anappropriate ratio, such as a 1:1 buffer:tissue ratio or a 2:1buffer:tissue ratio. In some embodiments, the extraction buffer may be50-100 mM Sodium Phosphate+2 mM EDTA+250 mM NaCl+0.1% Tween80, pH 8.5.Removal of chlorophyll and large cellular debris involves the use offiltration. Celpure300 is added at a ratio of 33 g/L and mixed for 15minutes. Feed pressure (<30 PSI), filtrate pore size (0.3 microns),clarifying agent (Celpure300) and biomass loaded per square meter ofmembrane surface are all controlled to ensure passage of the antigens.

Clarified extract is concentrated with a TFF system (such as theSartorius AG system). In some embodiments, the cassette pore size (fore.g., 30 kDa), an appropriate TMP as described herein, and load ofclarified extract per square meter of membrane surface area arecontrolled.

The clarified extract is concentrated and washed 7× with an appropriateion-exchange chromatography equilibration buffer (such as 50 mM SodiumPhosphate 75 mM NaCl, pH 6.5), The Capto Q ion-exchange column isequilibrated for five column volumes with 50 mM Sodium Phosphate+75 mMNaCl, pH 6.5, the feed is loaded, washed with equilibration buffer, andthe column eluted/stripped with high salt.

Antigen fractions are collected in the elution for preparation forCobalt IMAC chromatography. IMAC is equilibrated for five column volumeswith 50 mM Sodium Phosphate+500 mM Sodium Chloride, pH 8.0, feed isloaded, washed with equilibration buffer and eluted using imidazole.

The elution fraction is diluted to conductivity, pH is checked andloaded onto a multi-modal ceramic hydroxyapatite (CHT) chromatographycolumn. The CHT resin is equilibrated with five column volumes ofequilibration buffer (5 mM Sodium Phosphate, pH 6.5). Antigens areeluted using a gradient of phosphate and NaCl. Loading ratio, column bedheight, residence time and chromatography buffers are all controlled.Formulation and concentration of the antigens takes place using a TFFsystem (such as the Sartorius AG system). Pore size (in kDa), TMP, loadper square meter of membrane surface area and pore material are allcontrolled, as further discussed herein.

Antigen is next concentrated to a suitable concentration, such as 3mg/ml, and diafiltered with a suitable buffer (for example, phosphatebuffered saline, pH 7.4). Formulated antigen is sterilized and storedappropriately. In some embodiments, sterilization is provided via a PESfilter.

FIGS. 9, 10, and 11 illustrate the various steps of the antigenpurification platform according to multiple embodiments andalternatives. FIG. 9 shows the purity of the antigen product after theCapto Q chromatography step has concluded, FIG. 10 shows the purity ofthe antigen product after the affinity chromatography step, and FIG. 11shows the purity after the CHT chromatography column.

Examples 3, 4, 5, and 6—H5 rHA, H7 rhA, WNV rDIII, and LFV rGP1/2

As shown in FIG. 12 , the antigen purification platform according tomultiple embodiments and alternatives has successfully purified H5 rHA,H7 rhA, WNV rDIII, and LFV rGP1/2, FIG. 12 contains two images takenfrom the conclusion of the antigen purification platform: the image onthe left contains a SDS Page gel indicating purity for the viral vectorTMV NtK (where NtK is an abbreviation for N-terminal lysine) andinfluenza antigens, and the image on the right contains a western blotindicating the immunoreactivity for West Nile and Lassa Fever antigens.As shown by the clear and visible bands in FIG. 12 , each antigenproduct is highly pure. Therefore, the antigen purification platformaccording to multiple embodiments and alternatives consistently purifiedeach type of antigen on a commercial scale it was used with in a mannerthat is also compliant with cGMP regulations. In the same manner, thisplatform is expected to be reproducible to purify virtually any type (ifnot all types) of antigen.

Production of Recombinant Antigen—Virus Conjugates

Table 3 illustrates the steps of the conjugation of recombinant antigenaccording to multiple embodiments and alternatives.

TABLE 3 Production and Purification of Recombinant Antigen OperativeIn-Process In-Process Steps Unit Operations Controls Analytics  1Concentration/Diafiltration Pore Size, TMP, UV280 or BCA, of AntigenPore Material, SDSPage, pH, kg/m² Conductivity  2Concentration/Diafiltration Pore Size, TMP, UV260, SDSPage, of TMV1295.10 Pore Material, pH, Conductivity kg/m²  3 Formulation of EDCMixing, Weight Concentrate Check  4 Formulation of Sulfo-NHS Mixing,Weight Concentrate Check  5 Combine Antigen and Molar Ratio, pH,Conductivity, TMV 1295.10 Mixing, Volume SDSPage  6 Addition of EDC EDCMolarity, pH, Conductivity, Mixing, Volume SDSPage  7 Addition ofSulfo-NHS Sulfo-NHS pH, Conductivity, Molarity, Mixing SDSPage Volume  8Conjugation Reaction Time, Temperature, Mixing  9 Reaction QuenchingTime, Temperature, Mixing, Molarity of Amine Group 10 Diafiltration toRemove Pore Size, TMP, pH, Conductivity, Reactants Pore Material,SDSPage, kg/m² Reactants (EDC/NHS) 11 Concentration/Formulation PoreSize, TMP, Certificate of of Purified Vaccine (Drug Pore Material,Analysis Substance) kg/m²

In an embodiment, the steps of a conjugation platform are as follows:

Purified antigen and virus are separately concentrated and diafilteredinto a slightly acidic buffer, such as a 2-(N-morpholino) ethanesulfonicacid (MES) buffer containing NaCl.

A water soluble carbodiimide, such as 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide (known as EDC) is formulated in purified water to amolarity of 0.5 M.

A chemical reagent for converting carboxyl groups to amine reactiveN-hydroxysulfosuccinimide esters, such as ThermaoFisher's Sulfo-NHS, isformulated in purified water to a molarity of 0.1 M.

Antigen and virus are combined based upon weight or molarity and mixedto homogeneity (e.g. 1:1 mg:mg addition).

The freshly prepared water soluble carbodiimide (such as EDC) is addedto the mixture while mixing based upon molarity.

A chemical reagent for converting carboxyl groups to amine reactiveesters (such as Sulfo-NHS) is added based upon molarity within oneminute of EDC addition. The conjugation reaction begins and is continueduntil a predetermined mixing stop time, such as four hours, and the roomtemperature is controlled.

The reaction is quenched by adding free amines, and the chemical linker(for example EDC and Sulfo-NHS) is removed through a multi-modalchromatography step, such as Capto® Core 700, or diafiltration into aphosphate buffered saline. According to multiple embodiments andalternatives, the residual impurities are removed from the results ofthe conjugation reaction, sometimes referred to herein as a conjugatemixture, based on sized differences between impurities as the retentate,and the conjugate mixture as the permeate.

The conjugate mixture is diluted to target concentration. At this point,the virus-antigen conjugate is prepared for use as a purifiedvaccine/drug substance. A suitable delivery mechanism of the vaccinewould include a liquid vial or lyophilized material to be reconstitutedwith physiologic buffering for project injection. Injection could beintramuscular or sub-cutaneous. Other delivery methods are contemplated,including without limitation intra-nasal.

Example 7—Conjugation of H7 rHA to TMV

FIG. 13 provides an illustration of the conjugation of a recombinantantigen (denoted by the “vaccine antigen”) to a virus, with lighter- anddarker-shaded ovals representing the extent of conjugation for thevaccine antigen depicted in the example. The lighter shade representsfree virus, while the darker shade represents antigen conjugated to theprotein coat of the virus. Also, as indicated in FIG. 13 , some virusescontain coat positioned proteins around the RNA genome. For example, theviral vector TMV NtK includes N-terminal lysines that serve as connectorpoints to the coat proteins. In some embodiments, portions of the virusassociated with N-terminal lysine residues are modified to enhancepresentment for binding of recombinant antigen providing amine-targetedconjugation of the protein, for example antigen to virus. In connectionwith the discussion of radial measurement herein, the viral radiusgreatly increases following conjugation of the recombinant antigens tothe viral coat proteins. In some embodiments, modification is performedwhen enveloped viruses are changed to allow enhanced presentment oftheir residues.

As shown in FIGS. 14-20 , the conjugation platform of recombinantantigen to virus has successfully conjugated H7 rHA to TMV. FIGS. 14-16show an analysis based on sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (“SDS-PAGE”) of the conjugation between H7 rHA to TMV atpH 5.50. As illustrated in these figures, nearly all of the H7 rHA wasconjugated to the TMV within 2 hours. The disappearance of the rHAprotein band and simultaneous appearance of complexes staining above the200KDa marker indicates the complex formation. The reactivity of thebands with HA-specific antibodies further establishes this conclusion.

SEC-HPLC reports also indicated successful conjugation of H7 rHA to TMVin accordance with the current embodiments of the conjugation platform.FIG. 17 shows a SEC-HPLC report of free TMV product. In FIG. 17 , theSEC-HPLC report of the free TMV product produced the signal datadetailed in Table 4 below.

TABLE 4 SEC-HPLC Data of Free TMV RT Width Area Peak [min] [min] AreaHeight % Symmetry 13.233 0.77 1078.39 23.41 100 0.39

FIG. 18 shows a SEC-HPLC report after H7 rHA is conjugated to TMV forfifteen minutes according to current embodiments of the conjugationplatform. In FIG. 18 , the SEC-HPLC report after H7 rHA is conjugated toTMV for fifteen minutes produced the signal data detailed in Table 5.

TABLE 5 SEC-HPLC Data After H7 rHA is conjugated to TMV for 15 MinutesRT Width Area Peak [min] [min] Area Height % Symmetry 26.539 0.52 553.7517.65 100 0.83

FIG. 19 shows a SEC-HPLC report after H7 rHA is conjugated to TMV fortwo hours according to current embodiments of the conjugation platform.In FIG. 19 , the SEC-HPLC report taken after H7 rHA is conjugated to TMVfor two hours according to current embodiments of the conjugationplatform produced the signal data detailed in Table 6 below,

TABLE 6 SEC-HPLC Data After H7 rHA is conjugated to TMV for 2 Hours RTWidth Area Peak [min] [min] Area Height % Symmetry 13.304 0.73 37.300.86 0.36 0.43 20.569 1.83 167.16 1.52 1.59 0.00 22.336 1.17 62.55 0.890.59 0.64 24.489 2.05 73.35 0.60 0.70 1.34 26.510 0.54 10153.91 316.3096.56 0.80 29.649 0.83 21.16 0.42 0.20 2.15

As illustrated in FIGS. 19 and 20 , the SEC-HPLC reports indicated thatall TMV rods were coated with some H7 rhA after conjugation for fifteenminutes, and more H7 rhA was added to the rods for up to two hours.After two hours, no additional conjugation was detected. According tomultiple embodiments and alternatives, the SEC-HPLC reports indicatethat the conjugation reaction achieves at least about 50% reduction innon-conjugated, native molecular weight, virus coat protein, and thatapproximately 3% free TMV remained after conjugation took place for fourhours.

As illustrated in FIG. 20 , western blot analysis of the conjugateproduct indicated successful conjugation of H7 rhA to TMV via covalentattachment. FIG. 20 shows a western blot analysis of the various stepsof the conjugation platform according to current embodiments, whereinall samples were loaded at 10 μL. The various lanes illustrate differentconjugation reaction times between the antigen and the virus. Lanes 14and 13 show that all the TMV rods were coated with the antigen afterfifteen minutes. After two hours, lanes 6-9 illustrate that noadditional conjugation took place.

Example 8 Inactivation of TMV NtK

In order to avoid viral contamination of biopharmaceutical products, itis often necessary to inactivate (or sterilize) the virus to ensure thevirus is no longer infectious. In addition, many regulatory agencieshave enacted rules (such as the cGMP regulations) that require at leastone effective inactivation step in the purification process of viralproducts. While UV-C radiation has been used in water treatment systemsfor many years, its use with biopharmaceutical products remainsunexplored and there are limited studies regarding its ability toeffectively inactivate viruses.

Accordingly, following virus production and purification but prior toconjugation with recombinant antigen, various UV-C conditions (i.e.energy density and wavelength) and various TMV concentrations wereevaluated in order to effectively inactivate and sterilize TMV NtK.While many energy densities were tested, only the higher levels ofenergy densities successfully inactivated TMV NtK. In addition, it wasdetermined that successful virus inactivation is concentration dependentbecause when the TMV solution was not diluted to an appropriateconcentration, the UV-C irradiation did not effectively sterilize everyvirus in the sample. Therefore, the TMV solution must be appropriatelydiluted to permit the UV-C irradiation to interact with and effectivelyinactivate each virus.

As shown in FIG. 21 , various amounts of UV-C irradiation (with energydensities between 300 J/m² and 2400 J/m²) were tested on Nicotianatabacum plants to evaluate infectivity. As shown in FIG. 21 , thelesions were reduced to zero after an UV-C energy dosage of 2400 J/m²,therefore indicating successful inactivation of the virus. In addition,energy dosages at much higher levels were also tested, and it wasdetermined that successful inactivation of TMV NtK also occurred atenergy densities ranging between 4800 J/m² and 5142 J/m².

According to multiple embodiments and alternatives, the steps of theviral inactivation (following purification but before conjugation) areas follows:

Dilution of the TMV NtK solution to a concentration less than 50micrograms/ml, as measured by A260 (which is a common method ofquantifying nucleic acids by exposing a sample to UV light at awavelength of 260 nm and measuring the amount of light that passesthrough the sample).

0.45 micron filtration of the TMV solution to remove bacteria and anyother large species that might interfere with UV line of sight, andother purification steps according to present embodiments, occurimmediately before inactivation with UV light.

Inactivating the TMV NtK by exposing the virus to light in the UVspectrum with an energy density between about 2400 J/m² and about 5142J/m². In some embodiments, the energy density of the UV light is betweenabout 4800 J/m² and about 5142 J/m². According to multiple embodimentsand alternatives, the wavelength of the UV light is 254 nm.

Next, the inactivated TMV NtK is ready to be conjugated to therecombinant antigen.

These viral inactivation steps are designed for commercial scalabilityand compliance with the cGMP regulations

Example 9—pH Dependency of Conjugation

To evaluate whether incubating the virus at an acidic pH results in highquality conjugation, an experiment was performed using the same batchesof virus, antigen, buffers, and esters, but changing only theformulation of the virus. In reaction 1, TMV was formulated into 1× MESConjugation Buffer at pH 5.50 at a concentration of 3.1 mg/ml, accordingto multiple embodiments and alternatives. In reaction 2, TMV wasconcentrated to 11.0 mg/ml in phosphate buffer and added directly as 15%of the conjugation reaction volume. After these steps, the conjugationprocess was monitoring by SEC wherein an ordered decrease in free TMVfrom zero minutes (indicated by T=0) would indicate successfulconjugation.

As shown in Tables 7 and 8, reaction 1 exhibited successful conjugation(due to the ordered decrease in free TMV from zero minutes) whilereaction 2 was unsuccessful as shown by the percent remaining free TMV.

TABLE 7 Reaction 1, Successful Conjugation-TMV Formulated in Acidic pHReaction 1, Free (TMV TMV Formulated Peak Remaining in MES at Area by %Free Sample 3.1 mg/mL) SEC TMV Free 284.8 nm 11104 N/A MK T = 0   154.9nm 9732 100% T = 5   139.8 nm 3909  40% T = 15′ 142.8 nm 1815  19% T =30′ 149.4 nm 1039  11% T = 45′ 155.6 nm 769  8% T = 60′ 153.2 nm 777  8%

TABLE 8 Reaction 2, Unsuccessful Conjugation-TMV Formulated in PhosphateBuffer Reaction 2 (TMV at Free 11.0 mg/mL TMV in Peak RemainingPhosphate Area by % Free Sample Buffer) SEC TMV Free 64.2 nm 27590 N/ANtK T = 0   67.5 nm 14750 100% T = 5   68.8 nm 14916 101% T = 15′ 66.9nm 13046  88% T = 30′ 73.3 nm 11705  79% T = 45′ 75.8 nm 8109  55% T =60′ 80.0 nm 11020  75%

Accordingly, as shown in Table 7, incubation of the virus in acidic pHresults in a conjugation greater than 90%. If the acidic pH incubationstep does not occur, then the percent conjugation remains less than 50%(as shown in Table 8).

Based on this experiment, a model for conjugation (shown in FIG. 22 )was developed. According to multiple embodiments and alternatives,conjugation between purified virus and purified antigen (denoted by“rHA” in FIG. 22 ) is greatly enhanced by improving the chemicalreadiness of the virus to engage the antigen (referred to herein as“activating,” “activation,” or “activates”) by exposing the virus to aconjugation environment. In some embodiments, virus activation occurs byformulating the virus in an acidic pH prior to the conjugation reactionsuch that positive charge aggregates on the virus surface. In someembodiments, the activating step involves exposing the virus to a pH ofabout 5.5 or less for a period of time sufficient for activation. Insome embodiments, such period of exposure to the conjugation environmentis between about 18 and 72 hours. According to multiple embodiments andalternatives, processing the purified virus in an acidic pH activatesthe virus by charging the coat protein lysine. As a result of thisactivation step in the conjugation environment, positive chargesaggregate on the virus surface (as shown in FIG. 22 ) via the clusteringof the amine groups and the virus is ready for conjugation with thecarboxyl end of the recombinant antigen.

The virus activation steps, according to multiple embodiments andalternatives, are in contrast with traditional approaches in which thepH when storing viruses generally is maintained at or near neutral pH.As shown in FIG. 22 , the traditional approach does not aggregatepositive charge on the virus surface, and as a result the percentconjugation remains below 50% (see Table 8). Furthermore, theconventional approach utilizes phosphate buffers which promotesolubility at the expense of having favorable surface charge.

During the investigation of successful conjugations involving TMV, itwas observed that successful conjugations generally occurred when theDynamic Light Scattering (DLS)-measured radius of the virus increasedduring the activation step by at least a factor of 2.75 (see Table 9A,compared to Table 9B). In general, successful TMV conjugations (such asdiscussed with Table 9C) were characterized by an increase in DLS radiusfrom about 70 nm to about 195 nm or higher, as shown in these tables.

Based on the successful conjugation which utilized virus activation, aplatform was developed for conjugating purified antigen to purifiedvirus. According to multiple embodiments and alternatives, the steps forpreparing the purified antigen for conjugation are as follows:

To ensure pH control of the conjugation reaction, the purified antigenis formulated into a reaction buffer immediately prior to reactioninitiation.

Prior to conjugation, purified antigens are stored in phosphate bufferedsaline at neutral to slightly basic pH.

The antigen pH target typically is pH 5.50 to 6.50, depending upon thenature of the molecule.

To facilitate conjugation to the virus, the storage buffer is replacedwith a MES/NaCl buffer at acidic pH using ultrafiltration. The proteinconcentration is also increased to greater than 3 mg/mL.

The conjugation reaction is then initiated within four hours of antigenpreparation completion to prevent destabilizing the protein structure.

According to multiple embodiments and alternatives, the steps forpreparing the purified virus for conjugation are as follows:

After storage at neutral pH, the virus is activated at acidic pH priorto conjugation. For successful reactions, the virus is formulated fromphosphate buffer at pH 7.4 into acetate buffer at pH 5.50 for a minimumof about 18 hours to a maximum of about 72 hours prior to theconjugation reaction start. In some embodiments, the virus is formulatedfrom phosphate buffer at pH 7.4 into acetate buffer at pH 4.50 for aminimum of about 18 hours to a maximum of 72 hours prior to theconjugation reaction start. It was observed that storage of the virusfor greater than 72 hours at acidic pH creates self-association betweenthe viruses which causes virus insolubility and inhibits the efficiencyof the conjugation.

Tables 9A and 9B further demonstrate the activation step in terms ofincreasing the radius of the virus (in this case, TMV) as measured byDLS, Specifically, Table 9A provides data for DLS radius increase of TMVafter being activated, and before a successful conjugation occurred,with the antigens listed in the right-hand column. The “Factor by whichradius increased” divides the TMV radius after activation by the typicalTMV radius at neutral pH, which is about 70 nm, Conversely, Table 9Bprovides data for DLS radius increase of TMV after an activation stepwas started, in advance of unsuccessful attempts at conjugation, withthe antigens listed in the right-hand column. In Tables 9A and 9B, theleft column represents the standard radius of TMV rods at neutral pH andunder general storage conditions, i.e., before any activation occurs.

TABLE 9A Free TMV radii as measured by DLS (Prior to successfulconjugation) TMV radius after TMV radius at activation (nm) Factor bywhich neutral pH (DLS results) radius increased Antigen 70 nm 195.22.789 SG 70 nm 207.2 2.960 SG 70 nm 249.1 3.559 SG 70 nm 249.1 3.559 SG70 nm 228.6 3.266 SG 70 nm 234.1 3.344 SG 70 nm 234.1 3.344 SG 70 nm441.3 6.304 SG 70 nm 284.8 4.069 SG 70 nm 517.6 7.394 SG 70 nm 574.08.200 SG 70 nm 448.2 6.403 SG 70 nm 209.7 2.966 PH 70 nm 220.4 3.149 PH70 nm 495.6 7.080 PH 70 nm 517.6 7.394 PH 70 nm 266.8 3.811 CO 70 nm495.6 7.080 CO 70 nm 517.6 7.394 CO 70 nm 295.4 4.220 MI 70 nm 517.67.394 MI 70 nm 574.0 8.200 MI Average (nm): Average Factor 413.5 forIncrease: 5.176

TABLE 9B Free TMV radii as measured by DLS (Prior to unsuccessfulconjugation) TMV radius at TMV radius after neutral pH activation (nm)Factor by which (standard) (DLS results) radius increased Antigen 70 nm95.4 1.363 SG 70 nm 105.4 1.506 SG 70 nm 156.0 2.229 SG 70 nm 176.52.521 PH Average (nm) Average Factor 133.3 for Increase: 1.905

Following these preparation steps, the antigen and virus reactants weremixed to form a conjugate mixture and the conjugation progress wasmonitored using DLS and SDS-PAGE methods. Table 9C illustrates theaverage molecular radius of the conjugation reaction over time using DLSafter the virus was activated using acidic pH. As shown in Table 9C,molecular radius is one indicator of successful coating of the viralrods with antigen molecules.

TABLE 9C TMV NtK SEC and DLS History Soluble NTK SEC DLS Radius PeakArea (nm) 10750 496 9651 518 7106 574 5538 660

In turn, FIG. 23 shows an analysis based on the SDS-PAGE of theconjugation between the activated TMV NtK and purified antigen accordingto multiple embodiments and alternatives. As shown in FIG. 23 , theordered reduction in both free TMV NtK and free antigen over time,coupled with the appearance of protein bands of >200 kDA, indicatessuccessful conjugation.

Example 10—TEM Imaging of Different Ratios of Purified Virus to PurifiedAntigen for Conjugation

The desired conjugation reaction between purified virus and purifiedantigen is represented by the following formula:Virus+Antigen→Virus-Antigen  (Formula 1)

However, it is well known that antigens are prone to self-conjugationand the desired reaction may not be obtained, as shown by the followingformula:Virus+Antigen→Virus-Antigen+Antigen-Antigen  (Formula 2)

Self-conjugation of the purified antigen is a problem for the successfuldevelopment of vaccines because the antigen-antigen conjugates are notremoved during the size chromatography step and the result is aminimized or reduced immune response.

To address this self-conjugation problem, various experiments wereperformed to determine how to consume the unreacted antigens and antigenconjugates. First, the antigens were capped by exposing them to reagentsthat inhibited self-conjugation. While it was anticipated that thistraditional approach would be successful, this approach failed becausethe reaction occurred too quickly.

Next, the virus to antigen ratios were adjusted to determine suitableconjugation ratios. As shown in Tables 10 and 11 and FIGS. 24-30 , sevendifferent samples were analyzed by negative stain transmission electronmicroscopy (TEM) imaging. Samples 1-3 were control groups and samples4-7 contained different hemagglutinin (HA) to TMV ratios (at the mixingstep of the conjugation platform, as shown at operative step 5 of Table3).

TABLE 10 TEM Imaging Samples-Control Groups Apprx. Volume Temp. SampleDescription Lot (μl) Stored Concentration 1 HA Alone 19UL-SG-001 100 4°C. 1.01 mg/ml free HA 2 TMV NtK 18HA-NTK-001 100 4° C. 0.54 mg/ml Alonefree TMV NtK 3 HA:HA 19UL-SG-004 100 4° C. 2.335 mg/ml Conjugates withadded TMV NtK

TABLE 11 TEM Imaging Samples-Conjugates Approx. Volume Temp. SampleRatio Lot (μl) Stored Concentration 4 TMV:HA = 1:1  18KBP-VP-SG-002 1004° C.  5.2 mg/ml 5 TMV:HA = 1:1  19UL-SG-001 100 4° C. 1.688 mg/ml 6TMV:HA = 4:1  19UL-SG-002 100 4° C. 1.387 mg/ml 7 TMV:HA = 16:119UL-SG-003 100 4° C. 3.479 mg/ml

In the various designations listed herein, “KBP-VP” describes a TMVAntigen Presentation and is provided for reference purposes only. FIG.24 is a TEM image of sample 1 (free HA, lot 19UL-SG-001) at amagnification of 52,000× and a scale bar of 200 nm. In FIG. 24 , thissample contained small globular arrows (indicated by arrow A) andelongated particles (indicated by arrow B) that ranged from ˜5 nm to ˜9nm in size. The appearance of these particles shows a regular structureconsistent with ordered aggregation of HA in keeping with native trimerconformation. In addition, the particles were well dispersed withminimal instances of clumping.

FIG. 25 is a TEM image of sample 2 (TMV NtK alone, lot 18HA-NTK-001) ata magnification of 52,000× and a scale bar of 200 nm. In FIG. 25 ,rod-shaped particles (arrow A) were observed in sizes ranging from ˜125nm to ˜700 nm in length and ˜18 nm to ˜20.5 nm in width. Thesedimensions are consistent with the size and shape of TMV particles. Inaddition, a central ˜4 nm channel was observed in the rods (arrow B),which is a known characteristic of TMV. Multiple rods were frequentlyaligned parallel to their long axis and the surface of the rods weregenerally smooth. On a few occasions, small ˜8 nm to ˜10 nm globularparticles (arrow C) were observed both associated with the surface ofthe rods and not associated with the rod-shaped particles in thebackground. These globular particles (arrow C) did not resembleindividual HA trimers.

FIG. 26 is a TEM image of sample 3 (HA:HA Self-Conjugates with added.TMV NtK, lot 19UL-SG-004) at a magnification of 52,000× and a scale barof 200 nm. In FIG. 26 , rod-shaped particles were observed that rangedfrom ˜25 nm to ˜885 nm in length to ˜18 μm to ˜20.5 μm in width (arrowA) and a central ˜4 nm inner channel (arrow B). The rods were either notdecorated at all or sparsely decorated with small, proteinaceousparticles of various sizes and shapes arrow C). Some of the small,proteinaceous particles were also seen in the background, not associatedwith the rods (arrow D). FIG. 26 illustrates larger clumps of HAparticles, but the TMV looks identical to the unconjugated TMV (shown inFIG. 25 ) as expected.

FIG. 27 is a TEM image of sample 4 (TMV:HA in a 1:1 ratio, lot 18KBP-VP-SG-002) at a magnification of 52,000× and a scale bar of 200 nm.In FIG. 27 , rod-shaped particles were observed that ranged in size from˜50 nm to more than ˜1000 nm in length and ˜18 μm to ˜20.5 nm in width(arrow A) with a ˜4 nm central inner channel (arrow B). The particlerods were similar in size and shape to the conjugated. TMV observed inFIG. 28 , with the exception that the majority of the rods were heavilydecorated with small proteinaceous densities on their surface (arrow C).Some of the small, proteinaceous particles were also seen in thebackground, not associated with the rods (arrow D). The sample 5 shownin FIG. 27 looks superior to the other TEM images which is most likelydue to the difference in virus treatment prior to conjugation. For thisbatch, the virus was formulated at pH 5.50, then the pH was reduced to4.50 for 15 minutes, and brought back up to pH 5.50 at the start of theconjugation reaction. For the batches shown in FIGS. 28-30 , the viruswas formulated directly into pH 4.50 and held overnight before theconjugation.

FIG. 28 is a TEM image of sample 5 (TMV:HA in a 1:1 ratio, lot19UL-SG-001) at a magnification of 52,000× and a scale bar of 200 nm. InFIG. 28 , many rod-shaped particles were visible that ranged from ˜65 nmto ˜720 nm in length and ˜18 nm to ˜20.5 nm in width (arrow A) with a ˜4nm central inner channel (arrow B). The particle rods were similar insize and shape to the free TMV NtK (sample 2) observed in FIG. 25 .However, in contrast to the unconjugated virus shown in FIG. 25 , theparticle rods observed in FIG. 28 were moderately decorated withproteinaceous densities (arrow C). These densities were irregular inshape and size, and appeared to be randomly associated with the surfaceof the rods with no obvious pattern. Some of the small, proteinaceousparticles were also seen in the background, not associated with the rods(arrow D).

FIG. 29 is a TEM image of sample 6 (TMV:HA in a 4:1 ratio, lot19UL-SG-002) at a magnification of 52,000× and a scale bar of 200 nm. InFIG. 29 , rod-shaped particles were observed that ranged from ˜25 nm tomore than ˜1000 nm in length, and ˜18 nm to ˜20.5 nm in width (arrow A)with a ˜4 nm central inner channel (arrow B). The particle rods observedin FIG. 29 were similar in dimension to the previously conjugatedsamples, but the level of surface decoration of the small proteinaceousdensities (arrow C) ranged from moderate to sparse. Some of the small,proteinaceous particles were also seen in the background, not associatedwith the rods (arrow D).

FIG. 30 is a TEM image of sample 7 (TMV:HA in a 16:1 ratio, lot19UL-SG-003) at a magnification of 52,000× and a scale bar of 200 nm. InFIG. 30 , rod-shaped particles were observed that ranged in size from˜30 nm to more than ˜1000 nm in length and ˜18 nm to ˜20.5 nm in width(arrow A) with a ˜4 nm central inner channel (arrow B). The particlerods observed in FIG. 30 were similar in overall morphology to theprevious conjugated samples. However, the rods were only sparselydecorated with protein (arrow C) or not decorated at all. Only a fewsmall, proteinaceous particles were seen in the background, notassociated with the rods (arrow D).

FIGS. 24-30 illustrate that the 1:1 ratio exhibited full rod decoration,the 4:1 ratio exhibited moderate decoration, and the 16:1 ratioexhibited sparse decoration. Stated differently, the 1:1 ratio generatedvirus rods with heavy antigen decoration (i.e. more density) of HAantigen, while the 16:1 ratio generated viral rods with less antigendecoration (i.e. less density) of HA antigen on each rod. As a byproductof the conjugation reaction, HA-HA self-conjugates were observed,principally in the 1:1 ratio reactions. Furthermore, compared with the1:1 reactions, there appeared to be less free HA or HA-HA conjugates inthe 4:1 reaction and even less with the 16:1 reaction in TEM images aswell as SDS-PAGE reaction analyses (data not shown). In other words,there was higher conjugation efficiency of HA to solely TMV rods overallat the 16:1 ratio, but less density of HA per rod than the 1:1 reaction.

Example 11—Sedimentation Velocity Analysis of Different ConjugationConditions

Sedimentation velocity (“SV”), as measured in an analyticalultracentrifuge (“AUC”), is an ideal method for obtaining informationabout protein heterogeneity and the state of association of aggregation.Specifically, aggregates or different oligomers can be detected on thebasis of different sedimentation coefficients. This method also detectsaggregates or other minor components at a level below 1% by weight.Furthermore, SV provides high quality quantitation of the relativeamounts of species and provides accurate sedimentation coefficients forany aggregates.

In order to measure the amount of self-conjugated and unreacted HA, aswell as the amount of HA occupancy on TMV NtK with different conjugationconditions, the total signal associated with the sedimentation of freeantigen, free virus, and various TMV:HA ratios were measured usingSV-AUC. The following samples and descriptions are provided in Table 12:

TABLE 12 Samples and Descriptions for SV-AUC Sample Description LotConcentration 1 HA Alone 19S-G-001 1.01 mg/ml 2 TMV Ntk Alone18HA-NTK-001 0.54 mg/ml 3 TMV:HA = 1:1 19UL-SG-004  1.0 mg/ml 4 TMV:HA =1:1 18 KBP-VP-SG-  1.0 mg/ml 002 5 TMV:HA = 1:1 19UL-SG-001  0.8 mg/ml 6TMV:HA = 4:1 19UL-SG-002  1.0 mg/ml 7  TMV:HA = 16:1 19UL-SG-003  1.0mg/ml

These stocks were shipped cold (not frozen) and subsequently stored at2-8° C. until analyzed. 1×PBS from Corning was used for sample dilutionand as a reference blank. Sample 1 was diluted 1:1, and samples 2-7 werediluted 1:3 with 1×PBS to create the sedimentation velocity samples.These dilutions were carried out to bring the total absorbance of thesample within the linear range of the absorbance detection system.

Methods—The diluted samples were loaded into cells with 2-channelcharcoal-epon centerpieces with 12 mm optical pathlength. 1×PBS wasloaded into the reference channel of each cell. The loaded cells wereplaced into an analytical rotor, loaded into an analyticalultracentrifuge, and brought to 20° C. The rotor was then brought to3000 rpm and the samples were scanned (at 280 nm) to confirm proper cellloading. For samples 2-7, the rotor was brought to the final run speedof 9,000 rpm. Scans were recorded at this rotor speed as fast aspossible (every 3 min) for 11 hours (250 total scans for each sample).For sample 1 (the free HA), the rotor was brought to 35,000 rpm andscans were recorded every 4 min for 5.3 hours. The data was thenanalyzed using the c(s) method described in Schuck, P. (2000),“Size-distribution analysis of macromolecules by sedimentation velocityultracentrifugation and Lamm equation modeling,” Biophys. J. 78,1606-1619. Using this method, raw scans were directly fitted to derivethe distribution of sedimentation coefficients, while modeling theinfluence of diffusion on the data to enhance the resolution.

Results and Discussion—The high-resolution sedimentation coefficientdistributions for samples 1-7 are shown in FIGS. 31-37 . In thesefigures, the vertical axis provides the concentration and the horizontalaxis provides the separation on the basis of sedimentation coefficient.Each distribution has been normalized by setting the total area underthe curve to 1.0 (100%) to ensure the area under each peak provides thefraction of that species. Since samples 2-7 contain material sedimentingover a broad range of sedimentation coefficients, the data analysis hasbeen pushed to cover species sedimenting as fast as 2000 Svedburg units(S), and therefore the horizontal axis is on a log scale. To compensatefor the effect that log scaling could distort the visible area of thepeaks, the vertical axis has been multiplied by e sedimentationcoefficient, which correctly scales the relative peak areas. The datafor sample 1 (free HA) is presented traditionally using a linearsedimentation coefficient scale.

FIG. 31 is normalized sedimentation coefficient distribution for sample1 (HA alone, lot 195-G-001). Since free antigen is much smaller in sizethan virus, this sample was analyzed at a much faster rotor speed(35,000 rpm) than samples 2-7 (9,000 RPM) in order to adequatelycharacterize the size distribution. As shown in FIG. 31 , sample 1 issomewhat homogeneous, providing 73.7% main peak at 8.967 S. This was theexpected result for the HA antigen-only sample. This sedimentationcoefficient together with the width of the main boundary imply this mainpeak species has a molar mass of ˜222 kDa, which may indicate the mainpeak corresponds to roughly a HA trimer of the expected ˜70 kDa monomer.It is not physically possible for this sedimentation coefficient tocorrespond to monomer; instead, the main peak corresponds to anoligomeric state larger than monomer. As noted in Table 13 below, SECHPLC data at HA3 Singapore release, >90% of HA was identified in trimerstatus, with 3 of the 4 samples analyzed having greater than 50%trimerization.

TABLE 13 Extent of trimerization Pre-Clinical HA lot SEC SEC Antigen Lotnumber Trimer % Monomer % B/Colorado 18 KBP-VP 18HA-CO- 55.05% 44.95%CO-001 003 A/Michigan 18 KBP-VP- 18HA-MH- 11.93% 88.07% MH-002 007B/Phuket 18 KBP-VP - 18HA-PH- 84.51% 15.49% PH-002 003 A/Singapore 18KBP-VP - 18HA-SG- 94.52% 3.90% SG-002 003

As also shown in FIG. 31 , seven minor peaks sedimenting faster than themain peak were detected, which together represent 6.2% of the totalsedimenting absorbance. Presumably those two peaks represent productaggregates rather than high molecular weight impurities. The principalaggregate species at 12.4 S (4.25%) is sedimenting 1.4 times faster thanthe monomer, a ratio that falls within the range of 1.4 to 1.5 usuallyobserved for dimers. While that ratio suggests that this species is adimer of the main peak material (possibly a hexamer of the ˜70 kDamonomer), its sedimentation coefficient could also suggest that it is ahighly extended or partially-unfolded trimer of the main peak material(possibly a nonamer of the ˜70 kDa monomer).

In FIG. 31 , the next peak at 15.3 S (0.96%) is sedimenting 1.7× fasterthan monomer which suggests a trimer of the main peak material. Noabsorbance was detected for any sedimentation coefficients larger than30.9 S. Also, three minor peaks sedimenting more slowly than the mainpeak were also detected at 2.8 S (2.81%), 4.5 S (12.44%), and 6.0 S(4.94%). Of these minor peaks, the peak at 4.5 S most likely correspondsto antigen monomer.

FIG. 32 is the normalized sedimentation coefficient distribution forsample 2 (free TMV NtK, lot 18HA-NTK-001). As shown in FIG. 32 , nosedimenting material was detected below ˜60 S. This sample appearedquite heterogeneous, with the most abundant peak sedimenting at 229 S(30.9%). The second most abundant peak was detected at 191 S (28.7%). Itis not clear which peak corresponds to fully assembled virus. Inaddition, 25.3% of the total signal was observed sedimenting from 229 Sto 2,000 S, the largest sedimentation coefficient allowed in thisExample 11. It is unclear what the partially-resolved peaks from ˜60 Sto 2000 S represent.

FIGS. 33-37 show the normalized sedimentation coefficient distributionfor virus-antigen conjugates. Each of these figures shows a significantabsorbance of about 0.15 OD that did not sediment. This was establishedby increasing the rotor speed to 35,000 RPM after the completion of eachrun, in order to pelletize all remaining material. This material was notobserved in either the free antigen or the free TMV NtK samples.However, since this material did not sediment, it did not affect theresults of the measured size distributions.

FIG. 33 is the normalized sedimentation coefficient distribution forsample 3 (TMV to HA at 1:1 Ratio, lot 19UL-SG-004). As illustrated inFIG. 33 , the results in the sedimentation coefficient range from about40 S to 2000 S, and are similar to those observed for free virus (shownin FIG. 33 ). Three peaks were also observed in the sedimentationcoefficient range of 1-40 S: 9.9 S (28.3), 18.7 S (7.8%), and 34.5 S(1.0%). The peak observed at 9.9 S may correspond to the main peakobserved in the free HA sample (shown in FIG. 32 ). The variety ofsmaller peaks may reflect HA-HA self-conjugation events.

FIG. 34 is the normalized sedimentation coefficient distribution forsample 4 (TMV to HA at 1:1 Ratio, lot 18 KBP-VP-SG-002) and FIG. 35 isthe normalized sedimentation coefficient distribution for sample 5 (TMVto HA at 1:1 Ratio, lot 19UL-SG-001). The results shown in FIGS. 34 and35 are similar to those discussed for sample 3 (and shown in FIG. 33 ).However, some notable differences were observed, First, it is difficultto comment on differences observed for the free antigen sample (from1-40 S) because of poor resolution at this rotor speed. Nevertheless,FIGS. 34 and 35 show more total signal present from 40 S-2,000 S (whichis indicative of virus associated material) than sample 3.

FIG. 36 is the normalized sedimentation coefficient distribution forsample 6 (TMV to HA at a 4:1 ratio, lot 19UL-SG-002), and FIG. 37 is thenormalized sedimentation coefficient for sample 7 (TMV to HA at a 16:1ratio, lot 19UL-SG-003). FIG. 36 shows 91.1% total virus-associatedmaterial (i.e. virus-antigen conjugates) and FIG. 37 shows 99.4%virus-associated material (i.e. virus-antigen conjugates).

The results for the virus-antigen normalized sedimentation coefficientdistribution, as shown in FIGS. 33-37 , are set forth in Table 14. Aspreviously noted, the fraction between 1-40 S indicates the percent HAmonomer/trimer, and the fraction between 40-2000 S indicates the percentTMV NtK-HA conjugate, according to multiple embodiments andalternatives.

TABLE 14 SV-AUC Results of the Different Virus-Antigen ConjugatesFraction Between Fraction Between 40- 1-40 S (%) (HA 2000 S (%) (TMVSample Lot Ratio monomer/trimer) NtK-HA Conjugate) 3 19UL-SG-004 1:137.1 62.9 4 18 KBP-VP 1:1 26.1 73.9 SG-002 5 19UL-SG-001 1:1 31.4 68.6 619UL-SG-002 4:1 11.2 91.1 7 19UL-SG-003 16:1  0.6 99.4

The results in Table 14 indicate that a 1:1 ratio has moreself-conjugation of HA and HA products, as compared to the 4:1 and 16:1ratios. In addition, increasing the TMV:HA ratio results in virtuallycomplete engagement of HA products in TMV-conjugation events(approaching almost 100% conjugation in sample 7).

According to multiple embodiments and alternatives, decreasing theamount of HA in a conjugation reaction, by increasing the TMV NtK to HAratio from 1:1 to 16:1, results in: (1) reducing the aggregation of HAantigen on each TMV rod, as observed by Example 10 and FIGS. 24-30 ; (2)decreasing the amount of self-conjugation and unreacted HA events tonearly zero, as shown by FIGS. 31-37 and Table 14; and (3) increasingthe association of HA (as a percentage) to TMV compared withself-conjugation and unreacted HA events, as shown by FIGS. 31-37 andTable 14.

Example 12—Immune Response in Mice

To determine immune response following administration of the inventivevirus-antigen conjugates, mice were administered the conjugates asvaccines via intramuscular injection. Each vaccine was a TMV:HAconjugate produced at a 1:1 (TMV:HA) ratio as described herein,administered to most of the animals on Day 0 and 14 of the study(control animals were administered buffer alone, TMV alone, or HAalone). Those administered vaccine received either 15, 7.5, or 3.75 mcg(micrograms) of antigen, as shown below in Table 15. One cohort hadsamples drawn on Day 7, another at Days 14 and 21, and a third at Days28, 42, and 90, with the samples then subjected to hemagglutinationinhibition (HAI) assay.

Based on the assay, no measurable response from any animal for anyvaccine occurred at Days 7 or 14. However, initial responses were seenin some animals on Day 21. Specifically, 10/27 animals showed low levelresponses (only 1 of them >80 HAI titers) for H1N1 vaccine (InfluenzaA/Michigan/45/2015 (H1N1pdm09)). Also, 22/27 showed low level responses(only 2 of them >80) for H3N2 vaccine (InfluenzaA/Singapore/INFIMH-16-0019/2016). On Day 28, the number of animalswithin this cohort responding measurably to H1N1 vaccine was 8/29 with asingle animal at 80 HAI titers and all others less. For H3N2 vaccine,the number responding measurably was 14/29, also with a single animal at80 HAI titers and all others less.

The most pronounced results were observed from blood samples taken atDay 42 and Day 90, which are presented in Table 15, below. In thistable, a standard error of the mean (SEM) is provided with the averageand the fraction of animals responding (Fr.Resp.). It will be noted thatin each cohort, some of the mice received vaccines for Influenza Bviruses (B/Colorado/06/2017 (V) and B/Phuket/3073/2013 (Y),respectively). No response was detected in these animals on any of thedays, as expected because B-type influenza viruses and corresponding HAimmunogens are known to not generate HAI titers in mice with theefficiency and effectiveness as A-type HA immunogens.

TABLE 15 Immune response based on dose and time post-vaccination Day 42Day 90 Average HAI Titers Average HAI Titers Immunogen H1N1 Fr. RespH3N2 Fr. Resp H1N1 Fr. Resp H3N2 Fr. Resp 1. Vehicle 0 0 0 0 0 0 0 0alone 2.TMV alone: 0 0 0 0 0 0 0 0 15 mcg 3.HA Quad 15 0 0 0 0 0 0 0 0mcg 4.HA Quad 0 0 0 0 0 0 0 0 7.5 mcg 5.HA Quad 0 0 0 0 0 0 0 0 3.75 mcg6.V-HA Quad 20 ± 5.477 7/10 27 ± 7.218 8/10 274 ± 66.336 10/10 136 ±33.442 10/10 15 mcg 7.V-HA Quad 26 ± 4.733 9/10 22 ± 9.466 6/10 174 ±40.797  9/10 84 ± 45.77  6/10 7.5 mcg 8.V-HA Quad 19 ± 4.566 7/9  17 ±4.969 6/9  224 ± 62.993 8/9  40 ± 10.423 7/9 3.75 mcg

Separate from the previously described immune response study, and tofurther evaluate the inventive system in terms of suitable virus toantigen ratios, the humoral immune response in mice was evaluatedfollowing vaccination at various TMV:HA conjugate ratios (i.e., 1:1,4:1, 16:1) of both Influenza A Antigen and Influenza B Antigen alongwith controls as noted below. In this manner, various conjugation ratiosand their effect on immune response were studied. The mice receivingvaccination were administered 15 meg HA via injection on Day 0 and Day14 of the study, in a subcutaneous region dorsally. The serum antibodyresponses to the vaccination were then analyzed for HA-specificactivity. Tables 15 (H3 influenza virus used as capture protein) and 16(recombinant H3 protein used as capture protein) show the groupings ofmice (12 mice per grouping), and the agents that were administered, withthe right-hand column in each table presenting ELISA antibody (Ab)titers results.

TABLE 16 TMV:HA ratio study-A-type influenza HA. Conjugation ratioAverage ELISA Grouping Vaccine (TMV:Antigen) Ab Titer 1.Phosphate-buffered saline n/a  0 2. TMV-H3 H3 HA:HA  0 3. TMV-H3  1:1  04. TMV-H3  4:1 120 5. TMV-H3 16:1 200

FIG. 38 is a scatterplot associated with Table 16, which providesgraphical analysis of H3:HA Ab titers following administration ofvaccine at ratios of 0, 1:1, 4:1, and 16:1 (TMV:HA). FIG. 39 alsoillustrates graphically the results of geometric mean testing ofantigen-relevant Ab titers, using recombinant H3 antigen (Table 17) ascoating or capture H3 virus as capture protein (Table 17) that bindswith anti Influenza A H3 Antigen antibody. In terms of density (surfacearea of TMV occupied by HA), the trend for the three ratios progressesfrom 1:1 (most dense)>4:1>16:1 (least dense), as demonstrated by TEM andAUC analyses. In these figures representing ELBA results obtained withH3 antigen, the highest immune response was observed with the leastdense conjugate. That is, the trend for immune response was 16:1>4:1>1:1and went in reverse of the trend for density. Thus, surprisingly it wasfound at these ratios for TMV:HA, lesser density of conjugation tendedto provide better immune response. Possible explanations for thissurprising finding that antigenicity does not correlate with maximum HAconjugation events include: (1) more uniform antigen with less tono-unreacted or self-conjugated protein when the density iscomparatively lower; (2) there could be more efficient processing ofconjugated antigen and more preserved/uniform antigen conformation; and(3) the TMV rods (by way of example) may stimulate more antigenpresenting cells to migrate to the injection site and stimulateprocessing of attached antigen, or some combination of these factors.Note, however, that, just the presence of TMV particles does not replacethe need for conjugation (see, e.g., Tables 14 and 15).

In addition to Influenza A H3 Antigen, Influenza B Antigen also wasstudied (B-Phuket HA) using the binding propensity of recombinantInfluenza B Phuket Antigen and its corresponding antibody. Table 17,below, presents the results of this part of the study that was there isnot as clear of a showing of 16:1>4:1>1:1 based on the results ofaverage ELISA Ab titers.

TABLE 17 TMV:HA ratio study-B-type influenza HA. Conjugation ratioAverage ELISA Grouping Vaccine (TMV:Antigen) Ab Titer 1.Phosphate-buffered saline n/a 0 2. TMV-B B Phuket HA:HA 283± 3. TMV-B 1:1 211± 4 TMV-B  4:1  56± 5. TMV-B 16:1 329±

Even so, the 16:1 ratio demonstrated the highest average antibody titer.Thus, is reasonable to predict the same relationship between density andimmune response applies to the study of the Influenza B Antigen(B-Phuket HA). That is, as with the results of H3 antigen, immuneresponse will be higher for less dense forms of the conjugates.Additionally, there is reason to believe the conjugation reaction forthe 4:1 ratio did not proceed as the reactions for the other ratiosbecause of possible abnormalities during conjugation, and the fact thatneither electron microscopy nor ultracentrifugation analysis wereperformed on this sample. In any case, the data here show immuneresponse at all three ratios. The fact that immune response was achievedat multiple ratios underscores the robustness of the system for notbeing tied to any one particular ratio. This flexibility as seen withthe particular TMV-conjugated vaccines probably gives further indicationthat the system will work well both when other antigens are conjugatedto TMV besides the H3 and H1 antigens included in these studies, as wellas when other virus carriers besides TMV are used for the carrier.

In terms of clinical utility, a product conjugated in accordance withany of multiple embodiments and alternatives described herein may beutilized as a vaccine by delivering the purified antigen via a purifiedvirus, such as but not limited to the virus-antigen conjugates describedin Examples 7, 9, 10, 11, and 12. Still further, embodiments of thepresent disclosure include any vaccine products packaged in any numberof forms (e.g., vial) with appropriate buffers and additives, beingmanufactured from any virus-protein conjugate compositions, theconjugation of which is provided for herein. In this respect,embodiments include those wherein such vaccine products are amenable todelivery in the form of unit doses provided to a subject, such as butnot limited to administration by syringe or spray through routes thatinclude, but are not limited to, subcutaneous, intramuscular,intradermal administration, and nasal, as well as administration orallyby mouth and/or topically, to the extent clinically indicated. By way ofnon-limiting example, and without detracting from the breadth and scopeof the embodiments herein, the size of TMV (typically 18 nm×300 nm) andits rod-like shape promotes antigen uptake by antigen presenting cells(APCs), and thus serves to enhance immunity promoted by T cells (such asTh1 and Th2), including cellular responses, and to provide adjuvantactivity to surface conjugated subunit proteins. This activity is alsostimulated through viral RNA/URI interaction. As a result, the combinedeffect of vaccine uptake directly stimulates activation of the APCs.Humoral immunity is typically balanced between IgG1 and IgG2 subclassesthrough subcutaneous and intranasal delivery. Upon mucosal vaccinedelivery, responses also include substantial systemic and mucosal IgA.Cellular immunity is also very robust, inducing antigen-specificsecretion, similar to a live virus infection response. Whole antigenfusions allow for native cytotoxic T lymphocyte (CU) epitope processing,without concern for human leukocyte antigen (HLA) variance.

The broad (humoral and cellular) and augmented (amplitude andeffectiveness) immune responses associated with the multi-setpurification platform according to current embodiments are in sharpcontrast to subunit proteins tested without TMV conjugation, whichinduce little or no cellular or humoral immunity. The impact of theseimmune responses is that vaccines created via the multi-set platform,according to current embodiments, promotes highly protective responsesas single dose vaccines and offers speed and safety not offered by otherconventional vaccine platforms. Indeed, the conjugation platform isshown to work on a wide array of viruses and proteins (includingantigens), combined within a broad range of ratios and successfullyadministered at various doses, which again are indicative of therobustness of the system. Additional advantages of the multi-setplatform for producing vaccines in current embodiments include: aproactive antigen-stimulating approach for systemic immune protectionagainst pathogen challenge, the platform is highly adaptable to produceantigenic domains from disease pathogens (including virus glycoproteinsor non-secreted pathogen antigens), and the platform serves as anefficacious vaccine platform for both virus and bacterial pathogens.

In addition to advantages regarding vaccine applications, plant virusparticles purified via the multi-set platform according to currentembodiments can be formulated for various drug delivery purposes. Thesedifferent purposes may include: 1) immune therapy—through theconjugation of therapeutic antibodies to the surface of virus particlesand their delivery to enhance cytotoxic effect; 2) gene therapy—throughloading specific nucleic acids for introduction into particular celltypes for genetic modification, and 3) drug delivery—through loadingchemotherapeutic agents into virus particles for targeted tumordelivery.

As a brief example of the many advantages of the methods discussedherein, the multi-set platform according to multiple embodiments couldbe utilized as a drug delivery tool by first causing the purified virusto swell by exposing it to a pH shift as discussed above. Subsequently,the virus in this condition would be incubated with a solution ofconcentrated chemotherapeutic agent, such as doxorubicin, and the pH isthen reverted to neutral thereby causing the virus to return to itspre-swollen state and thereby entrapping the chemotherapeutic molecules.Next, the virus particle could be delivered to an organism by a deliverymechanism chosen from a group that includes, but is not necessarilylimited to, injection for targeted treatment of tumors.

Accordingly, the above descriptions offer multiple embodiments and anumber of alternative approaches for (i) the plant-based manufacture andpurification of viruses; (ii) the plant-based manufacture andpurification of antigens; and (iii) the formation of virus-antigenconjugates outside the plant that are therapeutically beneficial asvaccines and antigen carriers; and (iv) the delivery of therapeuticvaccines comprising a purified virus and purified antigen.

Example 13—Vaccine Stability Under Refrigerated and Room TemperatureConditions

Vaccines have dramatically improved human and animal health. Forinstance, in the 20^(th) Century alone, vaccines have eradicatedsmallpox, eliminated polio in the Americas; and controlled a variety ofdiseases throughout the world. However, vaccines are highly unstable andvery sensitive to changes in temperature. As discussed in Coenen et.al., Stability of influenza sub-unit vaccine. Does a couple of daysoutside the refrigerator matter? Vaccine 24 (2006), 525-531; influenzavaccines are generally unacceptable and inactive after five weeks atroom temperature storage (i.e. ˜25° C.). Of all the influenza vaccinesdiscussed in the F. Coenen article, only one vaccine exhibited stabilityfor 12 weeks at room temperature storage. This is a significant problemwith other vaccine types too. Accordingly, all current vaccines mustgenerally be refrigerated during the entire supply chain from the momentof commercial production until administration, often referred to as the“cold chain.”

While in a refrigerated environment, the majority of vaccines remainstable for the typical seventy-eight week goal of stability. However,the absolute requirement for cold chain is a global problem that haslimited the availability of vaccines worldwide because it is oftendifficult to guarantee in developing countries and has led to widespreadvaccine loss. Many efforts have been made to create room temperaturestable vaccines, but as discussed in the literature, those efforts havebeen unsuccessful. In addition, the cold chain is very costly tomaintain for manufacturers; as well as the doctors and organizationsreceiving, storing, and applying the vaccines to populations.Accordingly, there is a significant and global need for increasing thestability of vaccines and enhancing vaccine-antigen stability in orderto reduce the dependency on the cold chain and to ensure vaccines retaintheir potency until administration. In addition, improving stability canprolong the vaccine shelf life, which would facilitate the stockpilingof vaccines in the preparation of a potential pandemic and preventvaccine loss in unfavorable conditions. Along with other features andadvantages outlined herein, the scope of present embodiments meet theseand other needs. In doing so, the inventive purification and conjugationplatform extends the stability of protein-virus conjugates under bothrefrigerated and room temperature conditions.

There are several methods for determining antigen quality and vaccinestability including: (1) protein concentration s measured by BCA Proteinassay (which is based on the principle that proteins can reduce Cu²⁺ toCu⁺¹ in an alkaline solution which results in a purple color formation),(2) storage potency as measured by VaxArray® antibody array binding(which utilizes multiplexed sandwich immunoassays), (3) SDS-Page purityas measured in terms of a single migrating band, (4) pH as a measurementof the physical pollution properties, and when possible, (5) sizeexclusion chromatography to characterize the multimeric structure of theantigen. Moreover, a vaccine is considered unacceptable for use if itfails the BCA Protein assay, the VaxArray® test, or the SDS-Pageanalysis. In other words, if a vaccine fails any one of these threetests, the vaccine is unacceptable for use and inactive.

Accordingly, the five tests mentioned in the previous paragraph wereconducted on the following influenza HA antigens produced and purifiedin accordance with multiple embodiments and alternatives: H1N1(A/Michigan), H3N2 (A/Singapore), H1N1 (A/Brisbane), H3N2 (A/Kansas),B/Colorado, and B/Phuket. The following tables provide the stabilitydata and storage potency as measured at release and various times afterfilling into vials and stored under refrigerated conditions (4° to 8°C.). As used herein, an initial concentration or integrity refers to theconcentration or integrity of a compound, conjugate mixture,pharmaceutical product, vaccine, or the like at its release date, andthe release date is determined based on 21 C.F.R. Part 11 and ICH Q1AStability Testing of New Drug Substances and Products, Revision 2(November 2003), with the full contents of both being incorporated byreference herein.

TABLE 18 Stability of Purified H1NI (A/Michigan) Under RefrigeratedConditions Test Test Initial 1 3 4 5 6 Parameters Method Units (CoA)month months months months months Concentration BCA mg/mL 1.081 1.0571.068 1.066 1.060 0.921 Purity SDS PAGE %   97%  >99%   92%   88%   81%  76% Purity SEC Peak 1% 11.93% 6.18% 0.00% 2.83% 4.77% 4.92% Peak 2%88.07% 93.82%  100.00%  97.17%  95.23%  95.71%  Physical/ pH NA 7.4 7.47.4 7.2 7.3 7.3 Chemical Properties Storage VaxArray ® μg/mL 93 164 9871300 1085 1176 Potency

TABLE 19 Stability of Purified H3N2 (A/Singapore) Under RefrigeratedConditions Test Test Initial 1 3 4 5 6 Parameters Method Units (CoA)month months months months months Concentration BCA mg/mL 0.855 0.9000.891 0.908 0.885 0.795 Purity SDS PAGE %  >99%  >99%  >99%  >99%  >99% >99% Purity SEC Peak 1% 94.52%  97.95%  100.00%  100.00%  100.00% 100.00%  Peak 2% 3.90% 0.00% 0.00% 0.00% 0.00% 0.00% Physical/ pH NA 7.47.4 7.4 7.2 7.3 7.3 Chemical Properties Storage VaxArray ® μg/mL 746 6711037 624 872 1089 Potency

TABLE 20 Stability of H1N1 (A/Brisbane) Under Refrigerated ConditionsTest Test Initial 1 3 Parameters Method Units (CoA) month monthsConcentration BCA mg/mL 0.804 0.810 0.967 Purity SDS % >99% 78% 73% PAGEPurity SEC Trimer % 20.85% Trimer 11.21% Trimer 100% single Monomer79.15% 88.79% Monomer peak % Monomer Storage VaxArray ® μg/mL 1205 1064768 Potency

TABLE 21 Stability of H3N2 (A/Kansas) Under Refrigerated Conditions TestTest Initial 1 3 Parameters Method Units (CoA) month monthsConcentration BCA mg/mL 0.9 0.923 1.211 Purity SDS %  95%   93% 90% PAGEPurity SEC Trimer % 30.92% Trimer 5.20% Trimer 100% single Monomer %69.08% 94.80% peak Monomer Monomer Storage VaxArray ® μg/mL 916 10611094 Potency

TABLE 22 Stability of B/Colorado Under Refrigerated Conditions Test TestInitial 1 3 4 5 6 Parameters Method Units (CoA) month months monthsmonths months Concentration BCA mg/mL 0.848 0.855 0.862 0.873 0.8850.777 Purity SDS %   99%   63%   46%   40%   38%   35% PAGE Purity SECPeak 1% 55.05% 39.70% 38.87% 20.77% 20.88% 39.55% Peak 2% 44.95% 49.86%61.13% 79.23% 79.12% 60.45% Physical/ pH NA 7.3 7.5 7.4 7.3 7.3 7.4Chemical Properties Storage Vax Array ® μg/mL 541 446 733 528 823 1082Potency

TABLE 23 Stability of B/Phuket Under Refrigerated Conditions Test TestInitial 1 3 4 5 6 Parameters Method Units (CoA) month months monthsmonths months Concentration BCA mg/mL 0.957 0.895 0.912 0.951 0.8180.819 Purity SDS % 96.1%  >99%   97%   97%   93%   91% PAGE Purity SECPeak 1% 84.51% 90.05%  91.98%  85.96% 85.76% 92.47% Peak 2% 15.49% 9.95%8.02% 14.04% 14.24% 7.53% Physical/ pH NA 7.4 7.4 7.3 7.3 7.3 7.4Chemical Properties Storage VaxArray ® μg/mL 910 945 888 952 812 924Potency

Tables 18-23 illustrate that the purified free antigens exhibitdifferent patterns of stability. For instance, some antigens like H1N1(A/Michigan) and H3N2 (A/Singapore) appeared stable after 6 months withno significant deviations in measurements (as is typically observed).However, the other antigens such as B/Colorado and H1N1 (A/Brisbane),and to a lesser extent H3N2 (A/Kansas) and B/Phuket, exhibiteddegradation, loss of trimer, or loss of other key properties under theseconditions. For example, FIG. 40 is a SDS-PAGE analysis of purified.B/Phuket after 1 month under refrigerated conditions. In FIG. 40 , thedegradant bands of lower molecular weight below the intact band at ˜60kDA indicate that the purified B/Phuket antigen has degraded. Asexpected, the data in Tables 18-23 and FIG. 40 indicate that differentproteins exhibit different stabilities under refrigerated conditions.

When the same purified antigens are conjugated to TMV, according tomultiple embodiments and alternatives, the stability profile and storagepotency changes. In some embodiments, the inventive method enhances ameasure of stability of a conjugated compound comprising a protein andvirus particle, and includes activating the virus particle and thenmixing the virus particle and the antigen in a conjugation reaction toform a conjugate mixture, resulting in enhanced stability when theconjugated compound is placed in an unrefrigerated environment and aftera time period of at least 42 days following a release date. An exemplarystorage temperature is at least 20° C. The stability enhancement can begauged by comparing the stability of the conjugate mixture to that ofthe antigen alone. A suitable measure is any one or more of antigenconcentration, antigen integrity, or antigen potency. For example, whenthe measure of stability is antigen concentration, as measured by BCA orother appropriate methodology, a difference between concentration of theconjugated compound and concentration of the antigen alone of at least10% is within the scope of present embodiments. Likewise, when themeasure of stability is antigen integrity, as measured by SDS-PAGE,SEC-HPLC or other appropriate methodology, a difference betweenintegrity of the conjugated compound and integrity of the antigen aloneof at least 10% is within the scope of present embodiments. Likewise,when the measure of stability is antigen potency, as measured byantigen-antibody interaction based on ELISA results, or VaxArray®,surface plasmon resonance or other appropriate methodology, a differencebetween potency of the conjugated compound and potency of the antigenalone of at least 30% is within the scope of present embodiments.

Accordingly, the following tables provide the stability data of severalmonovalent formulations (at a TMV to antigen ratio of 1:1) at releaseand various times after filling into vials and stored under refrigeratedconditions (2° to 8° C.):

TABLE 24 Stability of the H1N1 (A/Michigan) to TMV Conjugate UnderRefrigerated Conditions Initial 1 3 6 Test Parameters Test Method (CoA)month months months Appearance Appearance Clear, Clear, Cloudy, Cloudy,Liquid Liquid Liquid Liquid Physical/Chemical pH 7.6 7.5 7.4 7.5Properties Protein BCA 0.898 1.066 1.101 0.994 Concentration Purity SDSPAGE >99.0 94.3 90.7 91.7 Storage VaxArray ® 325 329 415 208 PotencyAverage Size DLS 85.8 98.0 64.2 97.8 Radius Polydispersity 53.9 54.254.3 55.2

TABLE 25 Stability of the H3N2 (A/Singapore) to TMV Conjugate UnderRefrigerated Conditions Initial 1 3 6 Test Parameters Test Method (CoA)month months months Appearance Appearance Clear, Clear, Cloudy, Cloudy,Liquid Liquid Liquid Liquid Physical/Chemical Properties pH 7.6 7.4 7.47.5 Protein Concentration BCA 0.828 1.025 0.947 0.957 Purity SDSPAGE >99.0 94.9 92.8 92.9 Storage Potency VaxArray ® 363 496 468 500Average Size Radius DLS 72.1 86.3 77.8 71.1 Polydispersity 43 52.6 38.735.4

TABLE 26 Stability of the B/Phuket to TMV Conjugate Under RefrigeratedConditions Initial 1 3 6 Test Parameters Test Method (CoA) month monthsmonths Appearance Appearance Cloudy, Cloudy, Cloudy, Cloudy, LiquidLiquid Liquid Liquid Physical/Chemical Properties pH 7.6 7.5 7.4 7.5Protein Concentration BCA 0.874 1.010 0.995 0.940 Purity SDS PAGE >99.097.1 95.4 95.1 Storage Potency VaxArray ® 333 393 442 477 Average SizeRadius DLS 1040.7 1094.1 1428.2 1284.9 Polydispersity 47.5 42.1 49.653.3

TABLE 27 Stability of the B/Colorado to TMV Conjugate Under RefrigeratedConditions Initial 1 3 6 Test Parameters Test Method (CoA) month monthsmonths Appearance Appearance Cloudy, Cloudy, Cloudy, Cloudy, LiquidLiquid Liquid Liquid Physical/Chemical Properties pH 7.6 7.5 7.5 7.5Protein Concentration BCA 0.961 1.020 1.077 0.959 Purity SDS PAGE >99.096.0 96.0 94.9 Storage Potency VaxArray ® 218 653 599 585 Average SizeRadius DLS 2377.8 1025.7 1337.6 1153.9 Polydispersity 49.5 55.6 53.3≥57.1

In each of the conjugates described in Tables 24-27, the purity, pH,protein concentration, and storage potency is maintained through atleast six months of storage under refrigerated conditions. Further, thepolydiversity is also consistent over this timeframe. Polydiversityrefers to the variability of particle size in a complex product, andgenerally the lower the polydiversity the better the product.

In addition to the monovalent formulations, the following quadrivalentconjugate produced according to multiple embodiments and alternatives ata 1:1 TMV to antigen ratio exhibits strong stability under bothrefrigerated (4° to 8° C.) and room temperature (22° to 28° C.)conditions:

TABLE 28 Stability of the Quadrivalent Conjugate Under RefrigeratedConditions Test Test Initial 1 3 6 Parameters Method (CoA) month monthsmonths Appearance Appearance Cloudy, Liquid Cloudy, Liquid Cloudy,Liquid Cloudy, Liquid Physical/ pH 7.5 7.5 7.4 7.5 Chemical PropertiesProtein BCA 0.799 0.911 0.983 0.953 Concentration Identity VaxArray ®Antigen Antigen Antigen Antigen Binding Binding Binding Binding OccursOccurs Occurs Occurs Storage VaxArray ® A/Michigan: A/Michigan:A/Michigan: A/Michigan: Potency NtK = 123 NtK = 155 NtK = 103 NtK = 123μg/ml μg/ml μg/ml μg/ml a/Singapore: a/Singapore: a/Singapore:a/Singapore: NtK = 106 NtK = 110 NtK = 106 NtK = 101 μg/ml μg/ml μg/mlμg/ml B/Phuket: B/Phuket: B/Phuket: B/Phuket: NtK = 117 NtK = 140 NtK =114 NtK = 116 μg/ml μg/ml μg/ml μg/ml B/Colorado: B/Colorado:B/Colorado: B/Colorado: NtK = 78 NtK = 179 NtK = 134 NtK = 134 μg/mlμg/ml μg/ml μg/ml

TABLE 29A Stability of the Quadrivalent Conjugate Under Room TemperatureConditions Test Test Initial 2 1 2 Parameters Method (CoA) weeks monthmonths Appearance Appearance Cloudy, Cloudy, Cloudy, Cloudy, LiquidLiquid Liquid Liquid Physical/ pH 7.5 7.5 7.5 7.4 Chemical PropertiesProtein BCA 0.799 0.959 0.909 1.098 Concentration Identity VaxArray ®Antigen Antigen Antigen Antigen Binding Binding Binding Binding OccursOccurs Occurs Occurs Storage VaxArray ® A/Michigan: A/Michigan:A/Michigan: A/Michigan: Potency NtK = 123 NtK = 115 NtK = 126 NtK = 24μg/ml μg/ml μg/ml μg/ml a/Singapore: a/Singapore: a/Singapore:a/Singapore: NtK = 106 NtK = 108 NtK = 173 NtK = 29 μg/ml μg/ml μg/mlμg/ml B/Phuket: B/Phuket: B/Phuket: B/Phuket: NtK = 117 NtK = 96 NtK =84 NtK = 29 μg/ml μg/ml μg/ml μg/ml B/Colorado: B/Colorado: B/Colorado:B/Colorado: NtK = 78 NtK = 62 NtK = 124 NtK = 26 μg/ml μg/ml μg/ml μg/ml

TABLE 29B Stability of the Quadrivalent Conjugate Under Room TemperatureConditions Test Test Initial 3 6 Parameters Method (CoA) months monthsAppearance Appearance Cloudy, Liquid Cloudy, Liquid Cloudy, LiquidPhysical/ pH 7.5 7.4 7.5 Chemical Properties Protein BCA 0.799 0.9800.920 Concentration Identity VaxArray ® Antigen Binding Antigen BindingAntigen Binding Occurs Occurs Occurs Storage VaxArray ® A/Michigan:A/Michigan: A/Michigan: Potency NtK = 123 μg/ml NtK = 113 μg/ml NtK =114 μg/ml a/Singapore: a/Singapore: a/Singapore: NtK = 1.06 μg/ml NtK =115 μg/ml NtK = 80 μg/ml B/Phuket: B/Phuket: B/Phuket: NtK = 117 μg/mlNtK = 80 μg/ml NtK = 99 μg/ml B/Colorado: B/Colorado: B/Colorado: NtK =78 μg/ml MK = 139 μg/ml NtK = 120 μg/ml

Tables 28, 29A and 29B illustrate that the quadrivalent conjugateremains consistent and stable in terms of protein concentration, storagepotency, pH and appearance under both refrigerated and room temperatureconditions for at least six months. Table 30 provides the percent changein the storage potency of the various antigens described in Tables 29Aand 29B by comparing the initial potency to the storage potency at theparticular time.

TABLE 30 Percent Change in Storage Potency from the Initial Potency viaVaxArray ® 2 Weeks 1 month 2 months 3 months 6 months A/Michigan  93.50%102.44% 19.51%  91.87%  92.68% A/Singapore 101.89% 163.21% 27.36%108.49%  75.47% B/Phuket  82.05%  71.80% 24.79%  68.00%  84.62%B/Colorado  79.49% 158.97% 33.33% 178.00% 153.85%

Accordingly, as shown in Table 30, when the conjugate was placed in theunrefrigerated environment, the storage potency at the end of 30 dayswas at least 70% of the initial potency of the conjugate mixture withinthe first day post-conjugation. At the end of 90 days, the storagepotency of the conjugate mixture stored in the unrefrigeratedenvironment was at least 68% of the initial potency, and the storagepotency of the conjugate mixture was at least 75% at the end of at least180 days.

The following tables illustrate the stabilizing effect of theembodiments described herein by comparing the release conditions of thepurified recombinant antigen with the same protein conjugated to TMVaccording to multiple embodiments and alternatives. Furthermore,stability after six months under refrigerated conditions (4° to 8° C.)was compared between the purified antigen and the same antigenconjugated to TMV by analyzing the protein concentration, potency,SDS-page purity, and PH, as follows:

TABLE 31 Comparison Between the Stability of Purified B/Colorado Antigenand the B/Colorado to TMV Conjugate Colorado Release Data Colorado 6month Stability Free Conjugated Free Conjugated Assay Antigen (1:1)Antigen (1:1) BCA (mg/mL) 0.848 0.961 0.777 0.959 VaxArray ® 541 2181082 585 Potency (μg/mL) SDS PAGE 99 >99.0 35 94.9 Purity (%) pH 7.3 7.67.4 7.5

TABLE 32 Comparison Between the Stability of Purified B/Phuket Antigenand the B/Phuket to TMV Conjugate Phuket Release Data Phuket 6 monthStability Free Conjugated Free Conjugated Assay Antigen (1:1) Antigen(1:1) BCA (mg/mL) 0.957 0.874 0.819 0.940 VaxArray ® Potency 910 333 924447 (μg/mL) SDS PAGE Purity (%) 96.1 >99.0 91.0 95.1 pH 7.4 7.6 7.4 7.5

TABLE 33 Comparison Between the Stability of Purified H3N2 (A/Singapore)Antigen and the H3N2 (A/Singapore) to TMV Conjugate Singapore ReleaseData Singapore 6 month Stability Free Conjugated Free Conjugated AssayAntigen (1:1) Antigen (1:1) BCA (mg/mL) 0.855 0.828 0.795 0.957VaxArray ® 746 363 1089 500 Potency (μg/mL) SDS PAGE >99 >99.0 >99 92.9Purity (%) pH 7.4 7.6 7.3 7.5

TABLE 34 Comparison Between the Stability of Purified H1NI (A/Michigan)Antigen and the H1N1 (A/Michigan) to TMV Conjugate Michigan Release DataMichigan 6 month Stability Free Conjugated Free Conjugated Assay Antigen(1:1) Antigen (1:1) BCA (mg/mL) 1.081 0.898 0.921 0.994 VaxArray ® 93325 1176 208 Potency (μg/mL) SDS PAGE 97 >99.0 76 91.7 Purity (%) pH 7.47.6 7.3 7.5

Tables 31-34 illustrate the stability inducing properties of thepurification and conjugation embodiments, most clearly for theB/Colorado, B/Phuket, and H1N1 (A/Michigan) antigens in terms of puritymeasures. For the H3N2 (A/Singapore) and 9/Colorado antigens, thestability of the conjugate is also shown in terms of antigenconcentration. As shown in Tables 31-34, the purification andconjugation processes, according to multiple embodiments andalternatives, stabilized the antigen's physical properties, antigenicreactivity and other quantitative stability features.

Furthermore, Tables 29A, 29B, and 30 illustrate that the quadrivalentconjugate, produced according to multiple embodiments and alternatives,exhibits strong stability measures for at least six months, ortwenty-four weeks, at room temperature storage (22° to 28° C.). Comparedto conventional vaccines which exhibit an average stability of ˜5 weeksat room temperature (as discussed in the F. Coenen article mentionedabove), the vaccines according to multiple embodiments and alternativesexhibit stability for at least 5× greater than conventional influenzavaccines and several times longer than purified antigens. Accordingly,the formulation and conjugation processes according to multipleembodiments and alternatives stabilize extremely unstable antigens—suchas B/Colorado—and extend the stability of other antigens—such as H3N2(A/Singapore), H1N1 (A/Michigan), and B/Phuket—far beyond the stabilitylimits of free-antigens and conventional vaccines.

Example 14(a) to 14(h)—Quadrivalent Influenza Vaccine Studies

In order to demonstrate the safety, efficacy and utility of theembodiments disclosed herein for immunogenicity and protection againstseasonal virus challenge, several pre-clinical studies were conductedusing a quadrivalent seasonal vaccine candidate (referred to forpurposes of this example as “QIV vaccine”). The vaccine used in thestudies was manufactured in accordance with multiple embodiments andalternatives disclosed herein. The subject QIV vaccine contained thefollowing influenza HA antigens from the 2018/2019 North Americaseasonal influenza vaccine strains recommended by the World HealthOrganization, the Centers for Disease Control and Prevention, and theFDA's Vaccines and Related Biological Products Advisory Committee(VRBPAC), (A/Michigan/45/2015(H1N1)pdm09,A/Singapore/INFIMH-16-0019/2016 (H3N2), B/Phuket/3073/2013 (B Yamagatalineage), and B/Colorado/06/2017 (B Victoria lineage), conjugated toinactivated TMV NtK. In some embodiments, the four vaccine antigenconjugates, in a phosphate buffer solution with 0.01% thimerosal as apreservative (as a non-limiting example), are blended together to createa single injectable, quadrivalent vaccine formulation. As discussed inmore detail below, these studies demonstrated that the embodimentsdisclosed herein augment the immunogenicity of recombinant hemagglutininprotein antigens. This was determined by various measures and analyses,including hemagglutination inhibition and neutralizing antibody titers.Likewise, the studies described in this example indicate the QIVseasonal vaccine is immunogenic, as it provided a level of protection inall mammalian disease models tested to date from challenge with viralstrains homologous to the vaccine strain HA antigens. Except whereotherwise noted, the content of the inactivated TMV NtK and HAintermediates incorporated into the conjugation reaction were equal(1:1) on a mg:mg basis (i.e. weight (wt)) for the studies. Subsequentstudies with the QIV vaccine conjugated at an 8:1 TMV NtK:HA ratio (on amg:mg basis) of the drug substance intermediates, as a non-limitingexample, showed desirable humoral responses.

Table 35 provides an overview of the studies conducted on the QIVvaccine in accordance with the present example. “GLP” refers to GoodLaboratory Practices, such as the CPMP Note for Guidance on PreclinicalPharmacological and Toxicological Testing of Vaccines (CPMP/SWP/465/95)and the World Health Organization Guidelines on Non-Clinical Evaluationof Vaccines (WHO Technical Report Series, No. 927), with the fullcontents of both being incorporated by reference herein. Additionaldiscussion of various aspects of the studies follows the table.

TABLE 35 Overview of QIV Non-Clinical Studies for Example 14(a) to 14(h)Study Description Summary 14(a)-Evaluate Impact of QIV vaccine induced adose-dependent humoral immune Conjugation to TMV on response (HAItiters). Immunogenicity, and There were no adverse effects or injectionsite reactions Evaluate Durability of detected. Response Non-GLP, onmice 14(b)-Evaluate Impact of QIV vaccine induced a detectable humoralimmune Conjugation to TMV on response based on HAI and virusneutralization assays, Immunogenicity, and Titers were first detectableon Day 21 and were highest on Evaluate Durability of Day 90 postvaccination. Response There were no adverse effects or injection sitereactions Non-GLP, on mice detected. 14(c)-Immunogenicity QIV vaccineinduced a detectable humoral immune Challenge, response based on HAI andvirus neutralization assays Non-GLP, on ferrets against the fourantigens. The log reduction in viral titers observed in the QIVvaccinated ferrets challenged with A/Michigan/45/2015 (H1N1) wassuperior when compared to Fluzone ® and placebo. Equivalent logreduction in viral titers was observed in the QIV vaccinated ferrets andFluzone ® when compared to placebo in animals challenged withA/Singapore/INFIMH-16-0019/2016 (H3N2) virus. These data indicate thatthe QIV is immunogenic and provides a level of protection to influenzainfection in ferrets. There were no adverse effects or injection sitereactions detected. 14(d)-Matrix Immunization of mice with monovalentvaccine immunogenicity to conjugated to varying ratios (1:1 to 24:1 TMVEvaluate Desirable NtK:antigen) of TMV NtK scaffold. Formulation Ratio,Humoral immune responses indicated that a TMV Non-GLP, on mice NtK:HAratio of 8:1 (mg:mg) was a desirable formulation ratio for QIV14(e)-Pharmacokinetic: Study employed a monovalent vaccine conjugated toBiodistribution Using 1:1 inactivated or live TMV NtK carrier at a 1:1ratio of TMV Ratio of Virus to Antigen, NtK:antigen. Non-GLP; on rabbitsDistribution determined by quantitation of TMV NtK through a RT-qPCRmethodology, Peak TMV NtK values were measured at injection site muscle1 day post dosing and declined over the 7 day time course. Lymph nodesand spleen had relatively stable levels of TMV NtK vRNA genome copynumbers over the time course. Liver, heart, and testes had low levelsnear the limit of quantitation (LOQ) 1 day post-dosing that was belowLOQ over the rest of the time-course. Vaccine values were below the LOQin other tissues examined (blood, brain, lung, kidney and thymus). Thebiodistribution pattern of TMV NtK vRNA genome was consistent with bothlive and inactivated TMV NtK. No injection site reactions were detected.14(f) Pharmacokinetic: Study employed a vaccine conjugated toinactivated or Biodistribution Using 8:1 live TMV NtK carrier at a 8:1ratio of TMV NtK:HA Ratio of Virus to Antigen, antigen. Non-GLP, onrabbits RT-gPCR results consistently show that TMV vRNA copy numberpeaked on 1 day post-dosing in almost all organs except the injectionsite (muscle) which peaked at 3 days post-dosing and declinedthereafter. Biodistribution data also demonstrate a significant declineof residual vaccine after 1 day post-dosing in all of the organs testedexcept injection site (near or below the LOQ). Two immune organs (spleenand lymph nodes) had relatively stable levels of TMV vRNA genome copynumbers over the time course. 14(g) Safety: Intramuscular administrationof two doses of QIV (1:1 Repeat dose toxicity, TMV NtK:Antigen ratio) atdoses of 15 or 45 μg/HA GLP on rabbits once every four weeks was welltolerated No treatment-related or toxicologically significant clinicalfindings or inoculation site reactogenicity were observed. Notreatment-related or toxicologically significant effects were observedfor body weights, body weight changes, food consumption, bodytemperatures, ophthalmology, clinical chemistry, hematology, and organweights. A robust immunogenic response was also seen in rabbitsreceiving the low and high dose of the vaccine which was detected inmost animals on Study Days 42, 49, and 57. 14(h) Safety: Intramuscularadministration of two doses of QIV (45 μg/ Repeat dose toxicity, HA +1.44 mg TMV NtK) or TMV NtK (1.44 mg) carrier GLP on rabbits once everyfour weeks was well tolerated No treatment-related or toxicologicallysignificant clinical findings or inoculation site reactogenicity wereobserved. No treatment-related or toxicologically significant effectswere observed for body weights, body weight changes, food consumption,body temperatures, ophthalmology, clinical chemistry, hematology, andorgan weights. A robust immunogenic response was also seen in rabbitsreceiving the QIV vaccine which was detected in most animals on StudyDays 42, 49, and 57,

As summarized in Table 36, an immunogenicity study in BALB/c mice, usingmonovalent and quadrivalent preparations respectively, was conducted toevaluate a desirable formulation ratio, and to monitor for injectionsite reactions and clinical signs of toxicity.

TABLE 36 Immunogenicity in BALB/c Mice-Example 14(a) Antigen DoseVaccination Blood Collection Group N (μg) (Study Days) (Study Days) 1 50^(a ) 0, 14 0, 14, 28 Monovalent ^(b) 2 5 1.5 0, 14 0, 14, 28 3 5 30 0,14 0, 14, 28 Quadrivalent ^(c) 4 5 1.5 0, 14 0, 14, 28 5 5 7.5 0, 14 0,14, 28 6 5 15 0, 14 0, 14, 28 7 5 30 0, 14 0, 14, 28 ^(a)Phosphatebuffered saline only ^(b) Monovalent Vaccine:A/Singapore/INFIMH-16-0019/2016 (H3N2) ^(c) QIV Vaccine:A/Michigan/45/2015 (H1N1), A/Singapore/INFIMH-016-0019/2016 (H3N2),B/Colorado/06/2017 and B/Phuket/3073/2013

The study was conducted in the spirit of GLP regulations. BALB/c mice(N=5/group) were immunized on Day 0 and Day 14 with the respectivevaccine preparations. The animals were bled to prepare sera for HAIantibody titer analysis prior to dosing on Day 0 and Day 14, with afinal bleed collected on Day 28.

Table 37 (below) provides the number of animals that generateddetectable HAI titers, the titer range from positive animals, and thegeometric mean titer (GMT). The GMT value was calculated using thefollowing formula set forth in Armitage and Berry, Statistical Methodsin Medical Research, 2^(nd) Edition (1987), pp. 31-33, the full contentsof which are incorporated herein by reference:GM={x ₁ x ₂ x ₃ . . . x _(n)}^(l/n)  (Formula 3)

As shown in Table 37, prior to the first vaccination (Day 0), HAI titersfor sera samples from all study groups were below the limit of detection(<10). On Day 14 (prior to the second vaccination), antibody titers werebelow detectable levels in all groups except for the following: ⅕ micein Group 3 (30 μg dose of the monovalent vaccine) had an HAI titer of 10to A/Singapore/INFIMH-16-0019/2016 (H3N2) and ⅖ mice in Group 7 (30 μgdose of the quadrivalent vaccine) produced a titer of 10 toA/Singapore/INFIMH-16-0019/2016 (H3N2). In Table 37, HAI antibody titerswere the reciprocal of the highest dilution of sera that inhibitedhemagglutination by 4 HA units of virus.

TABLE 37 Immunogenicity in BALB/c Mice Results - Example 14(a) AntigenDay 14 Day 28 Group (μg) H1N1 ^(a) H3N2 ^(b) B/Vict ^(c) B/Yam ^(d) H1N1H3N2 B/Vic B/Yam 1 None 0/5 0/5 0/5 0/5 0/5 0/5 0/5 0/5 Monovalent(H3N2) 2 1.5 0/5 0/5 0/5 0/5 0/5 1/5 0/5 0/5    (40; 40) 3 30 0/5 1/50/5 0/5 0/5 5/5 0/5 0/5 (10; 10) ^(e) (10-160; 40)  QuadrivalentKBP-VP-V001 4 1.5 0/5 0/5 0/5 0/5 1/5 2/5 1/5 0/5     (20; 20)    (20;20) (20; 20) 5 7.5 0/5 0/5 0/5 0/5 5/5 4/5 1/5 0/5 (10-320; 53) (20-40;28) (40; 40) 6 15 0/5 0/5 0/5 0/5 4/5 2/5 2/5 0/5 (20-160; 95) (20-40;28) (10; 10) 7 30 0/5 2/5 0/5 0/5 5/5 5/5 2/5 0/5 (10; 10)   (160-640;279) (20-80; 52) (40; 40) ^(a) A/Michigan/45/2015 (H1N1pdm09); ^(b)A/Singapore/INFIMH-16-0019/2016 (H3N2); ^(c) B/Colorado/06/2017 (Vic)^(d) B/Phuket/3073/2013 (Yam) ^(e) Parenthetical data are the titerrange and geometric mean titer (GMT).

On day 14, HAI titers were detected against the H3N2 virus in the high(30 dose only in the monovalent and QIV vaccine groups. On Day 28, therewere dose-dependent increases in HAI titers against A/Michigan/45/2015(H1N1) in the quadrivalent vaccine and A/Singapore/INFIMH-16-0019/2016(H3N2) in both the monovalent and quadrivalent vaccines. HAI: titerswere detectable at antigen doses as low as 1.5 μg in a few animals withthe majority of animals generating antibody titers at 7.5 μg per HAantigen. No detectable titer was produced by the mice that had beenvaccinated with the monovalent vaccine to the other three strainstested. While less pronounced, the QIV vaccine also induced HAI titersin a subsect of mice against B/Colorado/06/2017. There were nodetectable HAI titers generated against the Yamagata lineageB/Phuket/3073/2013 component.

In sum, based on HAI assay data, the monovalent formulation vaccineinduced a detectable humoral immune response to the H3N2 virus. The QIVformulation induced a detectable humoral immune response against threeof the four antigens. The induced immune response against influenza H1N1and H3N2 antigens seen in mice vaccinated with the monovalent and QIVvaccine formulations was dose dependent. As shown in Table 37, the H1N1GMT ranged from 20 to 279 (increasing with the increasing dose) and theH3N2 GMT ranged from 20 to 52. In addition, no adverse clinical signs orinjection reactions were observed with the monovalent or QIVvaccinations.

As summarized in Table 38, an immunogenicity study in naïve mice (BALB/cmice) was conducted by assessing the immunogenicity of quadrivalentvaccines with and without TMV conjugation (1:1 ratio of TMV NtK:HAantigen), over time. The purpose of the study was to confirm the resultsfrom the prior study shown in Table 37, compare the immunogenicity ofQIV to a vaccine wherein the same antigens are not conjugated to the TMVNtK carrier, and analyze the durability of the immune response over 90days. The vaccines in the following table were conjugated at a 1:1 ratioof TMV NtK:HA antigen.

TABLE 38 QIV Vaccine Immunogenicity in BALB/c Mice - Example 14(b)Vaccine Blood Cohort Treatment Dose Antigen Vaccination Cohort Cohortcollections Necropsy Group Group N (μg) (days) Number N (days) Day 1Vehicle 28 None 0, 14 1 9 −1, 7 7 Control 2 9 −1, 14, 21 21 3 10 −1, 28,42, 90 90 2 TMV NtK 28 60 μg TMV NtK 0, 14 1 9 −1, 7 7 Control 2 9 −1,14, 21 21 3 10 −1, 28, 42, 90 90 3 HA Antigen 28 15 μg/HA 0, 14 1 9 −1,7 7 60 μg Total HA 2 9 −1, 14, 21 21 3 10 −1, 28, 42, 90 90 4 28 7.5μg/HA 0, 14 1 9 −1, 7 7 30 μg Total HA 2 9 −1, 14, 21 21 3 10 −1, 28,42, 90 90 5 28 3.75 μg/HA 0, 14 1 9 −1, 7 7 15 μg Total HA 2 9 −1, 14,21 21 3 10 −1, 28, 42, 90 90 6 Quadrivalent 28 15 μg/HA 0, 14 1 9 −1, 77 Vaccine 60 μg Total HA 2 9 −1, 14, 21 21 (referred to 60 μg TMV NtK 310 −1, 28, 42, 90 90 7 in FIG. 41 as 28 7.5 μg/HA 0, 14 1 9 −1, 7 7“KBP-VP 30 μg Total HA 2 9 −1, 14, 21 21 H1N1 and 30 μg TMV NtK 3 10 −1,28, 42, 90 90 8 KBP-VP 28 3.75 μg/HA 0, 14 1 9 −1, 7 7 H3N2”) 15 μgTotal HA 2 9 −1, 14, 21 21 15 μg TMV NtK 3 10 −1, 28, 42, 90 90

FIG. 41 shows the QIV vaccine induction of H1N1 and H3N2hemagglutination inhibition (HAI) titers in the mice over time. The dataincludes the geometric mean titer (GMT) of BALB/c mice dosed with 15 μgper HA of QIV vaccine. The GMT value was calculated using the followingformula:GM={x ₁ x ₂ x ₃ . . . x _(n)}^(l/n)  (Formula 3)

The mice with a titer value ≤10 were assigned a titer value of 5 for GMTcalculation, in accordance with the previously mentioned Armitage andBerry.

As shown in FIG. 41 , and Tables 39 and 40 below, HAI titers were onlyobserved in animals receiving the QIV vaccine (referred to in FIG. 41 as“KBP-VP HIM” and KBP-VP H3N2”) and only to the H1N1 and H3N3 antigens.There was no detectable immune response generated to the unconjugatedantigens. Titers were below detectable levels until Day 21 againstA/Michigan/45/2015 (H1N1) and A/Singapore/INFIMH-16-0019/2016 (H3N2).HAI titers remained detectable on Day 28 and Day 42 and were highest onDay 90. Moreover, the humoral response continued to increase for atleast 90 days, a feature that conventional influenza vaccines are notknown for. Since there was no detectable response with antigen alone,this test further supports the efficacy of conjugating the antigen tothe TMV NtK carrier in producing an immune response.

TABLE 39 Percentage of Animals and HAI Geometric Mean Titers GeneratedAgainst Influenza A Antigens - Days 21 and 28 Anti- Day 21 Day 28 genH1N1 ^(a) H3N2 ^(b) H1N1 H3N2 Group (μg) % GMT % GMT % GMT % GMT 1 None0 0 0 0 0 0 0 0 2 TMV 0 0 0 0 0 0 0 0 NtK 3 ^(c) 15 0 0 0 0 0 0 0 0 4^(c) 7.5 0 0 0 0 0 0 0 0 5 ^(c) 3.75 0 0 0 0 0 0 0 0 6 ^(d) 15 33% 11.7100% 29.4 30% 7.6 44% 10 7 ^(d) 7.5 10% 6.8 100% 25.2 30% 7.6 67% 12.6 8^(d) 3.75  0% 5  44% 10 22% 7.9 67% 10.3 ^(a) A/Michigan/45/2015 (H1N1)^(b) A/Singapore/INFIMH-16-0019/2016 (H3N2) ^(c) Quadrivalent vaccineantigens (A/Michigan/45/2015 (H1N1pdm09);A/Singapore/INFIMH-16-0019/2016 (H3N2); B/Colorado/06/2017 (Vict);B/Phuket/3073/2013 (Yam) alone, not conjugated to TMV NtK Carrier ^(d)QIV Vaccine (A/Michigan/45/2015 (H1N1pdm09);A/Singapore/INFIMH-16-0019/2016 (H3N2); B/Colorado/06/2017 (Vict);B/Phuket/3073/2013 (Yam) conjugated 1:1 TMV NtK:HA Antigen

TABLE 40 Percentage of Animals and HAI Geometric Mean Titers GeneratedAgainst Influenza A Antigens - Days 42 and 90 Day 42 Day 90 Antigen H1N1H3N2 H1N1 H3N2 Group (μg) % GMT % GMT % GMT % GMT 1 None 0 0 0 0 0 0 0 02 TMV 0 0 0 0 0 0 0 0 NtK 3 ^(c) 15 0 0 0 0 0 0 0 0 4 ^(c) 7.5 0 0 0 0 00 0 0 5 ^(c) 3.75 0 0 0 0 0 0 0 0 6 ^(d) 15 70% 15.2 80% 20 100%  183.8100%  91.9 7 ^(d) 7.5 90% 21.4 70% 21.4 90% 137.2 60% 26.4 8 ^(d) 3.7578% 15.9 67% 15.9 89% 117.6 78% 27.2 a. A/Michigan/45/2015 (H1N1) b.A/Singapore/INFIMH-16-0019/2016 (H3N2) ^(c) Quadrivalent vaccineantigens (A/Michigan/45/2015 (H1N1pdm09);A/Singapore/INFIMH-16-0019/2016 (H3N2); B/Colorado/06/2017 (Vict);B/Phuket/3073/2013 (Yam) alone, not conjugated to TMV NtK Carrier ^(d)QIV Vaccine (A/Michigan/45/2015 (H1N1pdm09);A/Singapore/INFIMH-16-0019/2016 (H3N2); B/Colorado/06/2017 (Vict);B/Phuket/3073/2013 (Yam) conjugated 1:1 TMV NtK:HA Antigen.

Like the HAI titers, virus neutralization (VN) titers were only observedin animals receiving the QIV vaccine. As presented in Table 41, VNtiters against A/Michigan/45/2015 (H1N1) andA/Singapore/INFIMH-16-0019/2016 (H3N2) were also not observed until Day21 VN titers remained detectable on Day 28 and Day 42 and were higheston Day 90.

TABLE 41 Neutralization Geometric Mean Antibody Titers Induced toInfluenza A Antigens A/Singapore/INFIMH- A/Michigan/45/2015 (H1N1)16-0019/2016 (H3N2) Dose μg/mL Dose μg/mL Day 30 15 7.5 Day 30 15 7.5 −150 50 50 −1 50 50 50 14 50 50 50 14 50 50 50 21 100 216 136 21 293.95158.74 79.37 28 216 151.6 114.9 28 147 114.87 141.42 42 216 186.6 233.342 370.35 151.57 186.61 90 635 857.4 606.3 90 864.05 303.14 263.9

In the study outlined in Table 38, the mice were also monitored forclinical signs and injection site reactions. At necropsy, organ weightswere measured and gross necropsy and histopathology on tissues wasperformed. There were no test article-related gross findings for anyanimals necropsied on Day 21 or Day 90.

For the animals euthanized on Day 21, a test article-related microscopicfinding of minimal to mild mixed cell infiltration at the injection sitewas noted in 3 out of 9 animals in Group 2 (TMV NtK Control), 1 out of 9animals in Group 3 (HA Alone), and 9 out of 9 animals in Group 6 (QIV).Minimal degeneration/regeneration of the myofiber of the injection sitewas also noted in 1 out of 9 animals examined from Group 6. No othermicroscopic test article related finding was noted for the miceeuthanized on Day 21 or Day 90.

In conclusion, the QIV vaccine induced a detectable humoral immuneresponse based on HAI and virus neutralization assays. The most robustresponse was to A/Michigan/45/2015 (FEINT) andA/Singapore/INFIMH-16-0019/2016 (H3N2). HAI and VN Titers were firstdetectable on Day 21 and were highest on Day 90. These data indicatethat the QIV vaccine is safe and immunogenic in mice.

Immunogenicity and Challenge Study in Ferrets—Example 14(c)

An immunogenicity and challenge study using the QIV vaccine was alsoconducted in ferrets, which are accepted as the most representativeanimal model for influenza infection, to evaluate vaccine efficacythrough reduction of viral loads post-challenge in relation to alicensed vaccine comparator. As shown in the study design illustrated inFIG. 42 , blood for immunogenicity was taken on the following studydays: - 3, 7, 14, 21, 28, and 42. On study day 43, the animals werechallenged and nasal wash for virus titer took place on study days 45and 47.

Briefly, ferrets N=30 (15M/15F) per group were immunized with the one ofthe following on Study Day 0 and 14:

1. Placebo buffer as negative control (same quantity as Group 4)

2. Fluzone® quadrivalent as a licensed comparator (15 μg per HA, 60 μgtotal HA) vaccine

3. QIV at 15 μg per HA antigen (60 μg total HA antigen; 60 μg TMV NtKcarrier)

4. QIV at 45 μg per HA antigen (180 μg total HA)

Following each dose, injections sites and clinical signs were monitoreddaily for 7 consecutive days. As shown in FIG. 42 , animals were bled atcertain intervals for measurement of HAI and neutralization antibodytiters. Animals from the individual dose groups were subdivided into twogroups (N=12, 6M/6F) and challenged on Day 43 with 1×10⁶ plaque formingunits (PFU) of either A/Michigan/45/2015 (H1N1)pdm09 orA/Singapore/INFIMH-16-0019/2016 (H3N2) (as non-limiting examples).Animals were monitored and nasal washes taken 2- and 4-dayspost-challenge to quantify residual influenza virus titers and assessviral clearance.

In ferrets dosed with either the 15 or 45 μg dose levels of QIV andFluzone®, hemagglutination inhibition (HI) antibodies were detectedagainst all four viruses tested [A/Michigan/45/2015 (H1N1)pdm09,A/Singapore/INFIMH-16-0019/2016 (H3N2), B/Colorado/06/2017 andB/Phuket/3073/2013], The QIV vaccine elicited the strongest HI responsesto B/Phuket, B/Colorado and A/Singapore/INFIMH-16-0019/2016 (H3N2). Theweakest HI responses were against the A/Michigan/45/2015 (H1N1) virus.Virus Neutralization (VN) titers were detected against all viruses, withorder of descending response: A/Michigan/45/2015 (H1N1)pdm09,A/Singapore/INFIMH-16-0019/2016 (H3N2), B/Phuket and B/Colorado/06/2017showing lowest response.

Following influenza challenge with A/Michigan/45/2015 (H1N1)pdm09, asshown in FIG. 42 , virus was detected in the nasal washes of allchallenged ferrets on Day 2 and 4 post challenge. FIG. 43 illustratesthe nasal wash titers following the H1N1 challenge (log 10 TCID₅₀/mL),wherein the QIV vaccine is referred to as “QIV”. As shown in FIG. 43 ,there were no statistical differences in viral titers at Day 2post-challenge, however, an increase in average body temperature of 1.0°C. was only observed in placebo controls. When compared to the placebogroup on 4-day post-challenge, all three vaccine groups yieldedstatistically reduced viral titers with the QIV vaccine showingdose-dependent log reductions (2.3 and 1.9 log 10 TCID50/mL) that werestatistically greater than Fluzone® (0.83 log 10 TCID50/mL). In FIG. 43, the p value is less than 0.05 for the Fluzone® value compared to theplacebo, and the p value is less than 0.001 for the QIV value comparedto Fluzone®. Also, the weight loss observed in Fluzone® and QIVvaccinated ferrets s comparable to placebo controls.

FIG. 44 illustrates the nasal wash titers following the H3N2 challenge(log 10 TCID₅₀/mL), wherein the QIV vaccine is referred to as “QIV”. Asshown in FIG. 44 , following challenge with A/Singapore/INFIMH-16-0019(H3N2), virus was detected in the nasal washes of all challenged ferretson Days 2 and 4 post-challenge. When compared to the placebo group, amaximal log reduction in viral titers was observed on Day 2 in the QIVvaccine 45 μg dose group (1.0 log 10 TCID₅₀/mL) which was statisticallylower than placebo. QIV vaccine and Fluzone® yielded log reductions of0.7 log 10 TCID50/mL and 0.5 log 10 TCID50/mL, respectively which werenot statistically reduced compared to the placebo group (the p value isless than 0.05). As shown in FIG. 44 , on Day 4 a comparable level ofsignificant log reduction in viral titers was observed for allvaccinated groups, Fluzone® (1 log 10 TCID₅₀/mL), QIV vaccine (15 μgdose, 1.4 log 10 TCID₅₀/mL), and QIV vaccine (45 μg, 1.2 log 10TCID50/mL). In addition, maximal average weight loss in the placebo andFluzone® vaccinated groups was comparable at 3.1% and 2.6%,respectively. QIV immunized animals had less infection-induced maximalaverage weight loss of 2.1% (15 μg QIV) and 1.2% (45 μg QIV).

In regards to studies of immunogenic response, the QIV vaccine of thepresent example induced a detectable humoral immune response against thefour antigens tested in HI and VN assays. A strong VN responsecorresponded with a reduction in nasal wash viral titers observed on Day4 post challenge in vaccinated ferrets challenged withA/Michigan/45/2015 (H1N1)pdm09. The log reduction in viral titersobserved on Day 4 in the QIV vaccinated ferrets was 1.1 to 1.5 loggreater when compared to Fluzone®. AgainstA/Singapore/INFIMH-16-0019/2016 (H3N2), a strong HI response wasobserved in all three groups of vaccinated ferrets. In ferretschallenged with A/Singapore/INFIMH-16-0019/2016 (H3N2), improvedprotection was observed for high dose group day 2 post infection and atday 4, with a similar log reduction in viral titers being observed inthe QIV vaccinated ferrets and Fluzone® both resulting in statisticallylower viral titers than Placebo. These data indicate that the QIVvaccine is immunogenic and provides a level of protection to ferretsagainst challenge with H1N1 and H3N2 homologous virus.

Matrix Immunogenicity Study in Mice—Example 14(d)

The goal of this study was to evaluate the immunogenicity of the QIVvaccine at different conjugation ratios of TMV NtK to HA antigen. Forthis study, a monovalent vaccine was formulated usingA/Singapore/INFIMH-16-0019/2016 (H3N2). As shown in Table 42, a fixeddose of HA with increasing TMV was compared to a fixed dose of TMV withdecreasing doses of HA (data not shown).

TABLE 42 Overview of Matrix Study Vaccine Treatment Antigen Dose TMV NtKVaccination Blood Collections Group Group N (μg) Content (days) (Days) 1Vehicle Control 5 0 0 0, 14 12, 28, 42 and 60 2 Monovalent 1:1 5 15 150, 14 12, 28, 42 and 60 3 Monovalent 4:1 5 15 60 0, 14 12, 28, 42 and 604 Monovalent 8:1 5 15 120 0, 14 12, 28, 42 and 60 5 Monovalent 16:1 5 15240 0, 14 12, 28, 42 and 60 6 Monovalent 24:1 5 15 360 0, 14 12, 28, 42and 60

Eight week old female BALB/c mice, five per group, were immunized withA/Singapore/INFIMH-16-0019/2016 (H3N2) monovalent influenza vaccine bysubcutaneous route of administration with the indicated vaccinecomposition on Days 1 and 14. Mice were bled and serum was collected onDays 12, 28, 42 and terminal serum was collected on Day 60.

FIG. 45 illustrates the immunogenicity of the monovalent vaccineproduced at varying ratios of TMV NtK: HA antigen. The relative IgGtiters are expressed as ng IgG per mL of serum. As shown in FIG. 45 , atday 60 (the end of the study), IgG titers induced by the monovalentvaccines prepared with increasing TMV-NtK: HA antigen conjugation ratioscontinued to show a significant improvement compared to the benchmark1:1 ratio group.

Furthermore, in the HA antigen study wherein the dose of HA was fixed at15 μg with increasing TMV NtK concentrations, the 8:1 and 16:1 vaccineformulations induced the highest mean response that was stable overtime. This data support the use of the vaccines produced according tomultiple embodiments and alternatives as an effective strategy togenerate humoral immunity.

Accordingly, the immunogenicity studies in mice and the efficacy studyin ferrets clearly demonstrate a significant increase in the generationof humoral immunity by the QIV vaccine of the present example.Additionally, immunogenicity was shown to be related to the conjugationof the HA antigen to the inactivated TMV NtK carrier. In mice, thehumoral response continued to increase over 90 days. Moreover,immunization of the QIV vaccine was able to significantly reduce H1N1and H3N2 viral loads following virus challenge that was greater than orequivalent to the conventional vaccine comparator, Fluzone®. In otherwords, vaccination with the QIV vaccine was able to reduce morbidity andvirus levels caused by infection with homologous H1N1 and H3N2 influenzastrains to the vaccine strain which were greater than or equal to theconventional vaccine comparator, Fluzone®. It is expected that vaccinesmanufactured in accordance with the present embodiments will showsimilar improved efficacy as the QIV vaccines of Example 14, across awide range of antigens conjugated to a virus such as TMV to be usedagainst many kinds of viruses.

Pharmacokinetic Studies—Examples 14(e) and 14(f)

Pharmacokinetic studies were performed to evaluate the biodistributionof the QIV vaccine following a single intramuscular injection in maleNew Zealand White rabbits over an eight day period. The study used aRT-qPCR methodology that was developed and qualified to measure the TMVNtK viral RNA extracted from tissue or blood, and the study compared twodifferent QIV vaccine formulations prepared with differing amounts ofthe TMV NtK carrier. The analyzed tissues included blood, skeletalmuscle (injection site), lymph nodes, spleen, thymus, heart, liver,lung, kidney and tested.

FIG. 46 shows the comparative distribution of the TMV vRNA in tissuesover time as measured by RT-PCR following a single injection of twodifferent QIV formulations, incorporating either 45 μg (1:1 formulationmonovalent vaccine) or 1440 μg (8:1 formulation quadrivalent vaccine) ofthe TMV NtK carrier in a 0.5 mL dose (as non-limiting examples). FIG. 47shows the biodistribution of the TMV vRNA in tissues by organ and dosegroup eight days after a single injection. In FIGS. 46 and 47 , “LOQ”refers to the limit of quantitation and “LOD” refers to the limit ofdetection.

As expected, the two formulations showed a consistent distributionpattern. In FIGS. 46 and 47 , the highest levels of the QIV werequantifiable from injection site skeletal muscle tissue through thestudy duration with amounts decreasing over time with both formulations.After skeletal muscle, the QIV was most abundant and detected throughoutthe study in spleen and draining lymph nodes, indicating that QIVtraffics to these tissues post-immunization. Transient levels weredetected in liver, heart, and testes at the 24 hour-post dosing timepoint and then below the LOQ with both formulations thereafter. The 8:1formulation had low, transient signal detected in blood, lung, andthymus tissues 24 hours post-immunization that were below the limit ofquantitation (LOQ) by Day 3 post-immunization, which is likely due tothe 32-fold higher amounts of TMV NtK included in that formulation. Noquantifiable QIV residuals were detected in brain or kidney tissue witheither QIV formulation.

Accordingly, in all formulations the vaccine was observed to trafficfrom the injection site to immune organs and no accumulation wasobserved in non-target organs. Likewise, the TMV-specific Q-RT-PCRsignal was observed to be dose dependent when measured at site ofinjection with rapid clearance from all non-target organs. Therefore,the detection in the immune system organs suggests a “depot” effect(i.e. sustained release of antigen at the site of injection) andprolonged stimulation of the immune system.

Toxicology Studies—Examples 14(g) and 14(h)

As previously noted in Table 35 and as described in Table 43 below,several toxicological studies were conducted according to GLPrequirements to evaluate the potential toxicity of the QIV vaccine andto support the clinical use of the QIV vaccine for the prevention ofdisease caused by infection with influenza virus (as a non-limitingexample). Two repeat dose toxicology studies in New Zealand Whiterabbits were performed using two formulations of QIV that differed inthe content of the TMV NtK carrier conjugated with the purifiedrecombinant HA antigens, according to multiple embodiments andalternatives. As described in more detail below, the studies revealed notreatment-related or toxicologically significant clinical findings whichsupport the safe use of the TMV NtK:HA conjugate in human studies.

TABLE 43 Overview of Toxicology Study Active Route of Study TreatmentComponents Administration Species Example Vehicle Control —Intramuscular New Zealand White Rabbis 14(g) QIV Low dose  60 μg HAIntramuscular New Zealand White Rabbis 1:1 QIV 15 μg/HA  60 μg TMV NtKformu- QIV High Dose 180 μg HA Intramuscular New Zealand White Rabbislation 45 μg/HA 180 μg TMV NtK Example Vehicle Control — IntramuscularNew Zealand White Rabbis 14(h) TMV NtK 1.44 mg TMV NtK Intramuscular NewZealand White Rabbis 8:1 QIV Control formu- QIV High Dose 180 μg /HAIntramuscular New Zealand White Rabbis lation 45 μg/HA 1.44 mg TMV NtK

As shown in Table 43, two repeat dose GLP toxicity studies wereperformed in rabbits using different virus to antigen ratios. In someembodiments, the QIV vaccine was administered as a single intramuscularinjection on an annual basis. To investigate the efficacy of thisapproach, a strategy of the number of human doses plus 1 (N+1) was usedwith the second dose administered 28 days following the first dose. Testarticles included the intended human high dose of the QIV vaccine ateach conjugation level of the TMV NtK carrier molecule (1:1 and 8:1formulations) and the high dose of the TMV NtK carrier alone.

In the repeat dose toxicity study of the QIV vaccine at a 1:1 Ratio ofTMV NtK carrier, HA Antigen, a seasonable influenza vaccine candidatewas administered intramuscularly twice (once daily) on study days 1 and29 to male and female New Zealand White rabbits, and the reversibilityof effects after a 28-day recovery period was evaluated. As shown inTable 44, each group consisted of 10 rabbits/sex/group. On study day 30(1 day after the last dose) five rabbits/sex/group were sacrificed andon study day 57 (28 days after the last dose) five rabbits/sex/groupwere sacrificed.

TABLE 44 Repeat Dose Toxicity Study Design, Example 14(g)-1:1 QIVformulation Vaccine No. of Rabbits Dose per Vaccine Dose (M + F) AntigenTMV NtK Necropsied on Group Treatment (μg) carrier (μg) Day 31 Day 57 1Control  0  0 5 + 5 5 + 5 (vehicle) 2 QIV 15  60 5 + 5 5 + 5 Vaccine 3QIV 45 180 5 + 5 5 + 5 Vaccine

The test articles were administered by intramuscular (IM) injection onstudy Days 1 and 29. At each injection, animals in each group received0.5 mL of the control article (Group 1), low dose vaccine (Group 2), orhigh dose vaccine (Group 3) using a 25-gauge needle attached to aplastic, 1-mL syringe (as non-limiting examples). The administrationsite was the relatively large muscle mass on the posterior aspect of thehind limb and was shaved or re-shaved (as appropriate). On each day ofinjection, the administration site was wiped with alcohol and allowed todry thoroughly for a minimum of 10 minutes prior to dosing. The IMadministration site alternated between hind limbs with the right hindlimb receiving the first dose.

Five rabbits/sex/group were euthanized on Study Day 31 (two days afterthe last dose), while the remaining study animals (4/sex/group)continued to be observed and were euthanized on study day 57 (28 daysafter the last dose). Experimental endpoints includedmorbidity/mortality; physical examinations, clinical signs of toxicity,and inoculation site (Draize) reactogenicity scoring; body weights; bodyweight changes; food consumption; body temperatures; ophthalmology;clinical pathology (clinical chemistry, hematology, coagulation); organweights; immunogenicity analysis; gross pathology at necropsy; andmicroscopic pathology.

All study rabbits survived to the scheduled necropsies. Notreatment-related or toxicologically significant clinical findings orinoculation site reactogenicity were observed. No treatment-related ortoxicologically significant effects were observed for body weights, bodyweight changes, food consumption, body temperatures, ophthalmology,clinical chemistry, hematology, and organ weights.

Fibrinogen levels were increased (p<0.05 or <0.01) in the vaccinetreatment groups usually at two days post dose. The increase infibrinogen in these groups was considered to be related to vaccinetreatment, but was considered an expected (inflammatory) responsefollowing treatment with an immunogenic substance. Fibrinogen was nolonger increased (p>0.05) at the end of the 28-day recovery period(reversible effect).

Mixed cell infiltration in the left sciatic nerve and injection site(side of last IM administration on Study Day 29) was a commonmicroscopic finding seen in the intramuscularly dosed vaccine toxicologystudies. This lesion was recoverable in the sciatic nerve, but not fullyrecovered in the injection site at the end of the 28-day recoveryperiod. This lesion was not considered adverse, but was an anticipatedfinding consistent with the administration of an immunogenic material.

In conclusion, intramuscular administration of the QIV vaccine at dosesof 15 or 45 μg once every four weeks for two injections (study days 1and 29) was well tolerated. Any findings noted did not result in anyadverse or limiting toxicity, were considered to be of minimaltoxicological significance noted in only one sex, reversible, transient,no alteration in organ function, etc.), and/or were anticipated findings(such as fibrinogen increases and mixed cell infiltration in theinjection site or sciatic nerve) following administration of animmunogenic substance.

Repeat Dose Toxicity Study—Example 14(h)

In the repeat dose toxicity study of the QIV vaccine at a 8:1 Ratio ofTMV NtK carrier, HA Antigen, a seasonable influenza vaccine candidatewas also administered intramuscularly twice (once daily) on study days 1and 29 to male and female New Zealand White rabbits, and thereversibility of effects after a 28-day recovery period was evaluated.As shown in Table 45, each group consisted of 8 rabbits/sex/group. Onstudy day 30 (1 day after the last dose) five rabbits/sex/group weresacrificed and on study day 57 (28 days after the last dose) fiverabbits/sex/group were sacrificed.

TABLE 45 Repeat Dose Toxicity Study Design, Example 14(h)-1:1 QIVformulation Vaccine Dose Vaccine Dose No. of Rabbits (M + F) per AntigenTMV NtK Necropsied on Group Treatment (μg) carrier (μg) Day 31 Day 57 1Control  0   0 4 + 4 4 + 4 (vehicle) 2 TMV NtK  0 1440 4 + 4 4 + 4Control 3 QIV Vaccine 45 1440 4 + 4 4 + 4

The test articles were administered by IM injection on Study Days 1 and29. At each injection, animals in each group received 0.5 mL of thecontrol article (Group 1), low dose vaccine (Group 2), or high dosevaccine (Group 3) using a 25-gauge needle attached to a plastic, 1-mLsyringe (as non-limiting examples). The administration site was therelatively large muscle mass on the posterior aspect of the hind limband was shaved or re-shaved (as appropriate). On each day of injection,the administration site was wiped with alcohol and allowed to drythoroughly for a minimum of 10 minutes prior to dosing. Theadministration site alternated between hind limbs with the right hindlimb receiving the first dose.

Four rabbits/sex/group were euthanized on study day 31 (two days afterthe last dose), while the remaining study animals (4/sex/group)continued to be observed and were euthanized on study day 57 (28 daysafter the last dose). Experimental endpoints includedmorbidity/mortality; physical examinations, clinical signs of toxicity,and inoculation site (Draize) reactogenicity scoring; body weights; bodyweight changes; food consumption; body temperatures; ophthalmology;clinical pathology (clinical chemistry, hematology, coagulation); organweights; immunogenicity analysis; gross pathology at necropsy; andmicroscopic pathology.

All study rabbits survived to the scheduled necropsies. Notreatment-related or toxicologically significant clinical findings orinoculation site reactogenicity were observed. No treatment-related ortoxicologically significant effects were observed for body weights, bodyweight changes, food consumption, body temperatures, ophthalmology,clinical chemistry, hematology, organ weights, and gross and microscopicpathology.

Fibrinogen levels were increased (p<0.01) in the treated groups usuallyat two days post dose. The increase in fibrinogen in these groups wasconsidered to be related to treatment, but was considered an expected(inflammatory) response following treatment with immunogenic substances.Fibrinogen was no longer increased (p>0.05) during the recovery period(reversible effect).

Accordingly, IM administration of the QIV vaccine candidate, orinactivated TMV NtK at doses of either 45 μg of each HA antigen (180 μgtotal HA±180 μg TMV NtK) or 1440 μg of each HA antigen, respectively,once every four weeks for two injections (Study Days 1 and 29) was welltolerated. The findings in these GLP toxicological studies did notresult in any adverse or limiting toxicity, were considered to be ofminimal toxicological significance (e.g., noted in only one sex,reversible, transient, no alteration in organ function, etc.), and/orwere anticipated findings (such as fibrinogen increases) followingadministration of immunogenic substances. No significant toxicologicalissues were noted with either 1:1 or 8:1 (TMV NtK:HA antigen)formulations of the QIV vaccines in these studies.

Furthermore, during the two GLP toxicological studies (Examples 14(g)and 14(h)), a robust antigen-specific immunogenic response was alsomeasured based on ELISA, HAI, and neutralization antibody titers inrabbits receiving the low and high dose of the QIV vaccine testarticles. FIG. 48 illustrates the total anti-HA IgG ELISA analysis fromrabbit serum samples following QIV vaccine immunizations on Day 1 andDay 29. As shown in FIG. 48 , the immunologic response peaked after thesecond administration of the QIV vaccine, as expected from theimmunization of naïve animals. Anti-influenza titers to all fourinfluenza antigens (Michigan H1N1, Singapore H3N2, B/Colorado, andB/Phuket as non-limiting examples) were measured in serum samples afterthe day 29 injection. Moreover, the mean anti-influenza titers up to90-fold higher (than the controls) were detected on study days 42, 49,and 57.

As shown in Table 46 below, HAI titers were detected in most animals onstudy days 42, 49, and 57 against A/Michigan/45/2015(H1N1),A/Singapore/MIN/H-16-0019/2019 (H3N2) and B/Colorado/06/2017 viruses (asnon-limiting examples). In contrast, HAI titers against theB/Phuket/3073/2013 virus (as a non-limiting example) were generally seenin 4 or fewer animals on Study Days 42, 49, and 57

TABLE 46 Percentage of Animals with Detectable HAI Titers on Study Day49 of the GLP Toxicology Studies Percentage of animals with detectableHI titer at Study Day 49 (HI Titer Response Range) A/MichiganH3/Singapore B/Colorado B/Phuket Dose (H1N1) (H3N2) (B/Victoria)(B/Yamagata) Placebo 0 0% 0% 0% 0% [from the 1:1 ratio study] Placebo 00% 0% 0% 0% [from the 8:1 ratio study] TMV NtK 1440 μg 0% 0% 0% 0% [fromthe 8:1 ratio study] QIV 15 μg/HA 100% 80% 50% 30% [1:1 TMV to (80-640)(20-160) (20-80) (10-20) HA Ratio] 45 μg/HA 100% 100% 70% 30% (80-640)(20-160) (20-80) (20-40) QIV 45 μg/HA 100% 100% 38% 100% [8:1 TMV to(40-320) (10-800) (10) (10-160) HA Ratio]

FIG. 49 illustrates the measurement of microneutralization GMT titersthroughout the GLP Toxicology Study using the 8:1 QIV Formulation foreach virus in the QIV vaccine. Also. Table 47 below, provides thepercentage of animals with detectable microneutralization titers onstudy day 49 of both GLP Toxicology Studies (i.e. both the 1:1 and 8:1QIV formulations). As shown in FIG. 49 and Table 47, neutralizationtiters against these same four viruses (as non-limning examples) werealso consistently measured in rabbits receiving the low and high dose ofthe vaccine on Study Days 42, 49, and 57, with the highest titers seenagainst the A/Michigan/45/2015 (H1N1)pdm09 andA/Singapore/INFIMH-16-0019/2019 (H3N2) viruses

TABLE 47 Percentage of Animals with Detectable MicroneutralizationTiters on Study Day 49 of the GLP Toxicology Studies Percentage ofAnimals with Measurable Titer A/Michigan H1N1 A/Singapore H3N2B/Colorado B/Phuket Study QIV QIV QIV QIV QIV QIV QIV QIV QIV QIV QIVQIV Time 1:1 1:1 8:1 1:1 1:1 8:1 1:1 1:1 8:1 1:1 1:1 8:1 Point 15 μg 45μg 45 μg 15 μg 45 μg 45 μg 15 μg 45 μg 45 μg 15 μg 45 μg 45 μg −2  0% 0%  0%  0%  0%  0% 0% 0% 0% 0% 0% 0% 14  0%  0%  0% 20% 40%  0% 0% 0%0% 0% 0% 0% 21  35%  30% 12.5%  60% 90% 12.5%  5% 5% 0% 0% 0% 0% 31  90% 75%  75% 55% 85% 12.5%  0% 0% 0% 0% 0% 0% 42 100% 100% 100% 100%  100%  88% 20%  56%  12.5%   100%  78%  100%  49 100% 100% 100% 100%  100% 100% 50%  75%  12.5%   80%  63%  100%  57 100% 100% 100% 80% 100%  100%0% 30%  0% 40%  50%  100% 

Accordingly, the studies discussed in this Example demonstrate that theQIV vaccine consistently produces a robust immune response followingintramuscular administration across three species (mice, ferrets andrabbits). The primary measure of immunity was the generation of HAIantibody titers, the recognized serum biomarker of protection forinfluenza infection. In immunologically naïve animals, the humoralimmune response was predominantly detected following a second (booster)immunization. Immunogenicity to the vaccine hemagglutinin antigens wasdependent upon conjugation to the TMV NtK carrier. The QIV vaccineprepared at a ratio of 8:1 TMV NtK carrier-to-recombinant HA antigen wasshown to be desirable. In a disease challenge model, immunization offerrets with QIV significantly reduced viral loads in animalssubsequently challenged with homologous H1N1 and H3N2 strains asassessed from nasal wash samples. This reduction in viral load wasgreater than or equal to that of a licensed comparator. Immunizationalso ameliorated clinical signs of morbidity associated with thechallenge viruses.

The studies also investigated the distribution and safety of the QIVvaccine. There has been no detection of edema or injection sitereactions in any of the studies. Biodistribution studies (measuring RNAfrom the TMV NtK carrier) with QIV found that, outside of the injectionsite muscle, QIV was measured in the spleen and lymph nodes at all timepoints tested indicating that TMV viral RNA was relatively stable ordecreased slowly in these organs which provides a potential mechanism ofaction and presentation of the antigens to the immune system. In repeatdose toxicity studies, the only finding was a reversible elevation infibrinogen levels from the clinical chemistry profile in the treatedgroups usually at two days post-dosing, an expected finding for animmunological substance. No other treatment-related or toxicologicallysignificant effects were observed for body weights, body weight changes,food consumption, body temperatures, ophthalmology, clinical chemistry,hematology, organ weights, and gross and microscopic pathology.

In conclusion, the data support the advancement of the TMV NtK conjugateto human clinical studies and offers clear advantages over currentlylicensed influenza vaccines. Since no toxicologically significantfindings have been observed with the QIV vaccine, the probability isincreased that no such events will be observed for any other antigen(s),purified in accordance with the antigen platform, that is conjugated tothe TMV NtK carrier described herein. Accordingly, it is expected thatother antigens conjugated to the inactive TMV NtK will have similarbiodistribution and toxicology profiles, and thus are suitable for usein humans.

Example 15(a) to (c)—Coronavirus Vaccine Candidate: RBD-Fc (SARS-2)Conjugated to TMV

A Covid-19 vaccine was produced by forming an antigen through theexpression of a recombinant version of the RBD SARS-2 spike proteinfused to the Fc domain of a human IgG1 in Nicotiana benthamiana (Nb)plants (as a non-limiting example). Before conjugation, the formedantigen was then purified according to an antigen purification platformas described herein, in accordance with multiple embodiments andalternatives, and the TMV virus particles were purified and inactivatedaccording to a virus purification platform described herein, inaccordance with multiple embodiments and alternatives. The purifiedrecombinant RBD-Fc antigen was then conjugated to the purified andinactivated TMV virus particle in accordance with the teachings ofmultiple embodiments and alternatives herein. Upon delivery to amammalian subject (e.g., human or animal), the RBD-Fc to TMV conjugatepresents the SARS-2 spike glycoprotein RBD fused to the human IgG1 Fcdomain via the chemical conjugation to the TMV virus particles. Asdiscussed in more detail below, the presentation of the RBD-Fc fusion inthis embodiment has been demonstrated to enhance Th1 and Th2 responsesin all mammalian disease models tested to date.

For this example, the antigen for the Covid-19 vaccine was selected bytargeting the RBD domain of the SARS-2 spike glycoprotein because itserves as the binding site for the human ACE-2 receptor and the bindingsite overlaps with characterized neutralizing antibodies. The SARS-2spike glycoprotein is found in the S1 subunit at the amino acidsnumbered approximately 320 to 520, In FIG. 50(A), the SARS-2 spiketrimer is shown in space-filling model with RBD circled in lateral andvertical views, FIG. 50(B) shows the RBD domain fused to a human 171allotype IgG1 Fc domain, expressed and purified from plants according tomultiple embodiments and alternatives.

Several considerations went into selecting SARS-2 RBD as a fusionpartner for developing the RBD-Fc antigen described in the presentexample and the next example. SARS-2 RBD is a binding site forneutralizing antibodies. Also, as discussed below, CR 3022 is a humanmAb isolated from a SARS patient that binds a domain in the SARS-2 RBDdomain. CR 3022 binding can neutralize both SARS-1 and SARS-2 CoV. TheSARS-2 RBD also presents the ACE-2 (angiotensin II) binding domain.

In accordance with the method for the present example, multigenicconstructs were designed and built to contain genes encoding theproteins necessary to synthesize a Covid-19 antigen targeting the RBDdomain of SARS-2. In the present example, the following antigen sequence(collectively referred to herein as the “RBD-Fc 121 Construct”) was usedfor the synthesis of a Covid-19 antigen:

1. “(SEQ ID NO: 1)” Signal Peptide: MGKMASLFATFLVVLVSLSLASESSA2. “(SEQ ID NO: 2)” SARS-2 virus Spike amino acids numbered 331-632:NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCSFGGVSVITPGTNTSNQVAVLYQDVNCTEVPVAIHADQLTPT 3. “(SEQ ID NO: 3”) Fc Hinge:VEPKSCDKTHTCPPCP 4. “(SEQ ID NO: 4”) IgG1 171 allotype Fc:APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

Illustratively, and without limitation, FIG. 51 shows an assembled TRBOexpression plasmid for a SARS-CoV2 RBD-Fc fusion peptide (i.e., antigen)for production in Nb plants. Successful infiltration and plantincubation were followed by extraction and purification of the antigenas described herein. As illustrated and discussed further herein, afusion peptide was formed having a first peptide which comprises areceptor binding domain (RBD) of a pathogen (specifically, a coronavirusand namely the SARS-CoV-2 spike (S) glycoprotein RBD), fused to a secondpeptide which comprises a fragment crystallizable (Fe) region of anantibody (containing a sequence of amino acids as set forth in SEQ IDNO: 4, human IgG1 Fe domain), and a hinge portion linking the firstpeptide and second peptide (containing a sequence of amino acids as setforth in SEQ ID NO: 3). In Example 15, the RBD contained a sequence ofamino acids as set forth in SEQ ID NO: 2, and in Example 16, the RBDcontained a sequence of amino acids as set forth in SEQ ID NO: 8. Itwill be noted that the reference to codons in the plasmid of FIG. 51includes both the RBD and Fe region as well as the hinge portion. Thesequences encoding the RBD-Fc antigen were optimized for efficient plantexpression. In accordance with the plasmid design, donor plasmidscontaining the referenced nucleic acid sequences for the fusion peptidewere synthesized. The antigen donor plasmid and TRBO expression plasmidwere digested using suitable restriction enzymes. The antigen wasligated into the TRBO expression plasmid and subsequent colonies werescreened to confirm clones. Assembled expression plasmids (FIG. 51 )from successful ligations were amplified in E. coli and DNA purified.The vector DNA-confirmed clones of the antigen in the TRBO expressionplasmid were used to transform Agrobacterium tumefaciens in preparationfor infiltration into Nb plants. Individual colonies were transferred ina sterile manner, incubated, and 20% glycerol stocks from culture wereproduced and retained as antigen Master Cell Banks. As furtherillustrated in FIG. 51 , the exemplary construct included a cauliflowermosaic virus (CaMV) 35S promoter, which is a DNA dependent RNA promoterset to transcribe the TMV expression vector with an accurate 5′ end. Inaddition, the TMV replicase is composed of the 126 kDa and 183 kDareplication associated proteins. In FIG. 51 , the “30 k” refers to amovement protein produced from a sub-genomic promoter in the 3′ end ofthe 183 kDa protein coding region. In the exemplary construct, theantigen gene is transcribed from a sub-genomic promoter in the 3′ end ofthe 30 kDa protein coding region and the 3′ untranslated region has aribozyme to insure termination of the transcript near the authentic 3′end. Furthermore, there is an E. coli origin for replication and borderregions for the TI plasmid and other elements for efficient replicationin Agrobacterium and insertion into plant cells.

Accordingly, a plasmid providing a construct for an antigen which isconjugable with a virus can be manufactured in accordance with multipleembodiments and alternatives herein. Such a construct may comprise firstand second coding regions that encode a fusion peptide. In non-limitingfashion, the first coding region contains a nucleic acid sequenceencoding an amino acid sequence as set forth in SEQ ID NO: 2 above, orby way of further example SEQ ID NO: 8 below. The first peptide maycomprise or substantially comprise a receptor binding domain of apathogen, such as a virus, non-limiting examples of which includecoronaviruses and influenza viruses as discussed further herein.Additionally, and again in similar non-limiting fashion, the secondcoding region contains a nucleic acid sequence encoding an amino acidsequence as set forth in SEQ ID NO: 4 above. The second peptide may be afragment crystallizable (Fe) region of an antibody capable of binding toa Fc receptor. In some embodiments, the first peptide and the secondpeptide are linked by a hinge portion coded by a third coding region.The third coding region may contain a nucleic acid sequence encoding anamino acid sequence as set forth in SEQ ID NO: 3 above. In someembodiments, the hinge portion is a portion of the Fe region. In someembodiments, one or more nucleic acid sequences identified herein willbe part of a heterologous expression system.

Plant Expression, Purification, and Characterization—Example 15(a)

in the present example, an RBD-FC 121 fusion peptide (hereafter,referred to in this example as “RBD-Fc 121 antigen”) was expressed intobacco plants, harvested then purified. Expression occurred in naïvewild-type Nb plants, which were infected with an expression vector (suchas the vector shown in FIG. 51 as a non-limiting example) for proteinreplication of the RBD-Fe 121 antigen, in accordance with multipleembodiments and alternatives herein, with growth and incubation of theplants occurring in a contained and controlled indoor environment.Purification of the RBD Fc-121 antigen was performed according to anantigen purification platform described herein (for example, asillustrated at Table 2 and FIG. 8 ), with the multi-modal ceramichydroxyapatite (CHT) chromatography column step omitted for thisparticular example. Accordingly, for the present example, FIG. 50(B)shows the RBD domain fused to the human 171 allotype IgG1 Fe domain,expressed and purified from plants according to multiple embodiments andalternatives.

As shown in FIGS. 52-53 , an antigen purification platform according tomultiple embodiments and alternatives successfully purified the RBD-Fc121 antigen resulting in high yields (>400 mg of RBD-Fc protein) withpure, stable antigen as RBD-Fc monomer in a manner that is compliantwith GLP regulations. FIG. 52 , taken from the conclusion of thepurification platform applied to this antigen, contains a SUS page gelindicating purity for the RBD-Fc 121 antigen, wherein lane 1 shows themarker and lane 2 shows the purified RBD-Fc 121 protein. As shown by theclear and visible bands in lane 2 of FIG. 52 , the RBD-Fc 121 antigenproduct is pure. In addition, the protein migration shown in FIG. 52 isconsistent with the SEC-HPLC report of the free, purified RBD-Fc 121shown in FIG. 53 . In FIG. 53 , the SEC-HPLC report of the free RBD-Fc121 antigen produced the signal data detailed in Table 48 below.

TABLE 48 SEC-HPLC Data of free, purified RBD-Fc 121 Antigen RT WidthArea Peak [min] [min] Area Height % Symmetry 13.428 0.64 327.78 7.1911.18 0.71 15.964 0.57 2605.08 68.46 88.82 0.68

Table 48 and FIG. 53 illustrate that >90′%© of the purified RBD-Fc 121antigen is in monomeric form. Likewise, low impurities were present inthe batches at less than 0.442 EU/mg. Therefore, the RBD-Fc 121 antigenwas successfully and sufficiently purified consistent with GLP.

The stability of the RBD-Fc 121 antigen is reflected in FIG. 54 andTable 49 (below). FIG. 54 contains a SDS Page gel of the purified RBD-Fc121 antigen 5 weeks after purification. In FIG. 54 , the lanes include:lane 1—marker, lane 2—blank, lane 3—blank, and lanes 4-6—RED-Fc 121antigen 5 weeks after purification. The clear and visible bands in FIG.54 indicate the purified RBD-Fc 121 antigen is highly stable. Inaddition, as shown in Table 49 below, the purified RBD-Fc 121 antigenmaintained its potency for at least 5 weeks following purification basedon ELISA results.

TABLE 49 Potency of Purified RBD-Fc 121 Antigen as Measured by ELISATest Parameter Test Method units 2 week 3 week 4 week 5 week PotencyELISA ug/mL 7010 8188 7096 5178

Conjugation and Preparation—Example 15(a)

The purified RBD-Fc 121 antigen was then conjugated with a TMV NtKcarrier. The TMV NtK virions with surface lysine residues for efficientconjugation were manufactured in Nb plants (again, as a non-limitingexample). The TMV NtK carrier was purified according to a viruspurification platform described herein (for example, as illustrated atTable 1 and FIG. 1 ). In accordance with multiple embodiments andalternatives herein, following purification the TMV NtK was subject tomicron filtration (e.g. 0.45) and treated with UV inactivation (asdiscussed in Example 8 herein).

The purified and inactive TMV NtK was then chemically conjugated to theRBD-Fc 121 antigen to produce a Covid-19 vaccine, in accordance withmultiple embodiments and alternatives herein (for example, asillustrated at Table 3), As shown in FIG. 55 , an exemplary conjugationprocedure may include at least the following steps:

Purified RBD-Fc 121 antigen was diafiltered into a 50 mM MES bufferedsalt (50 mM NaCl) solution, concentrated to a target concentrationappropriate for conjugation, and subjected to 0.2 micron filtration.

Purified RBD-Fc 121 antigen was conjugated to the purified TMV NtKparticles using EDC and Sulfo-NHS chemistries within a 1-hour mixingreaction.

As previously indicated, conjugation may occur at a range of TMVNtK:antigen (mg:mg) ratios, including without limitation 8:1, 4:1, and1:1.

Now returning to the present example, the RBD-Fc 121 antigen and TMV NtKunderwent a conjugation reaction performed at a pH of about 6.0, in EDC(4 mM) and Sulfo NHS (5 mM). In this regard, the of the conjugationreaction and the pH of the purification step need not be the same. Theconjugation reaction was quenched with free amine (Tris Base),optionally using a 30 kDa UF membrane to remove residual EDC, Sulfo NHS,and Tris Base.

Accordingly, the conjugated drug substance was diafiltered andformulated. As the conjugate exceeded 0.2 microns, all steps downstreamof the TMV NtK UV treatment and antigen 0.2-micron filtration weremaintained in a state of asepsis.

At this point, the TMV NtK:RBD-Fc conjugate will be (and in the case ofExample 15, was) ready for drug substance filling and drug productfilling. A suitable delivery mechanism of the vaccine would include aliquid vial or lyophilized material to be reconstituted with physiologicbuffering for project injection, with administration of the vaccineaccording to optional methods described herein.

To determine the percent conjugation between the TMV NtK and the RBD-Fc121 antigen, the SV was measured using an AUC. As previously noted, thefraction between 1-40 S indicates the percent RBD-Fc monomer/trimer, andthe fraction between 40-2000 S indicates the percent TMV NtK:RBD-Fcconjugate, according to multiple embodiments and alternatives.

In the present example, FIG. 56 shows the normalized sedimentationcoefficient for sample 4 (in which the TMV:RBD-Fc conjugate was dilutedby a factor of 28), and FIG. 57 shows the normalized sedimentationcoefficient for another sample (where the TMV:RBD-Fc conjugate wasdiluted by a factor of 10). FIG. 56 shows 100% total virus associatedmaterial (i.e. virus-antigen conjugates) and FIG. 57 shows 99.7% totalvirus associated material (i.e. virus-antigen conjugates). The resultsin FIGS. 56 and 57 indicate virtually complete engagement of the RBD-Fcproducts in TMV-conjugation events.

The successful conjugation between the TMV NtK and the RBD-Fc 121antigen was confirmed by SDS-Page analysis. FIG. 58 contains a SDS Pagegel of the TMV NtK:RBD-Fc conjugate at different time periodspost-conjugation. In FIG. 58 , the lanes include: lane 1—marker, lane2—the conjugate mixture at 0 min, lane 3—the conjugate mixture at 5 minpost-conjugation, lane 4—the conjugate mixture at 15 minpost-conjugation, lane 5—the conjugate mixture at 30 minpost-conjugation, lane 6—the conjugate mixture at 45 minpost-conjugation, lane 7—the conjugate mixture at 60 minpost-conjugation, lane 8 the final conjugate mixture after mixing, lane9—a TMV:HA conjugate as a control, lane 10—the conjugate mixture 0 min,lane 11—the conjugate mixture at 5 min post-conjugation, lane 12—theconjugate mixture at 15 min post-conjugation, lane 13—the conjugatemixture at 30 min post-conjugation, lane 14—the conjugate mixture at 45rain post-conjugation, lane 15—the conjugate mixture at 60 minpost-conjugation, lane 16—the final conjugate mixture after mixing, andlane 17—a TMV:HA conjugate as a control. The clear and visible bands inlanes 8 and 16, which are similar to the bands in lanes 9 and 17,indicate successful conjugation of TMV NtK to the RBD-Fc 121 antigen.

Accordingly, almost 100% conjugation of the RBD-Fc 121 antigen to the TMNtK was confirmed by AUC and SDS Page.

In Vitro Testing—Example 15(b)

To determine the efficacy of the TMV NtK to RBD-Fc 121 conjugate as aCovid-19 vaccine candidate, the binding of the antigen to CR 3022 andthe ACE-2 receptor were analyzed, as well as the immune response inmice. As discussed in more detail below, the data indicates the RBD-Fcantigen that was conjugated to TMV NtK, bound successfully to the humanACE-2 receptor. As further shown in Table 51, the initial immuneresponses stimulated after one vaccine dose with the TMV NtK:RBD-Fcconjugate supported this observation. Likewise, the RBD-Fc 121 antigenwhich was conjugated to TMV NtK SARS-RBD-specific, human neutralizingmonoclonal antibody (mAb), CR 3022.

To determine the binding efficacy of the conjugate, an ELISA test wasdeveloped to measure the RBD potency for both free antigen (i.e. RBD-FC121 alone) and conjugated forms (TMV to RBD-Fc) using CR3022 mAb forcapture. The ELISA test was conducted to find sera that did not bind tothe Fe portion of the antigen in order to eliminate background binding.This test was conducted through various methods including pre-absorptionand/or binding to the kappa region of the CR3022 light chain. FIG. 59illustrates the CR 3022 ELISA data by showing the conservation of RBDconformation among the free antigen and the TMV:RBD-Fc conjugateformulated at a 8:1 and 4:1 (TMV NtK:antigen) ratio, respectively. InFIG. 59(A), the ELISA standard curve illustrates the sensitivity andlinearity for binding of SARS-2 Spike antigen to the CR 3022 antibody.In FIG. 59(B), the TMV:RBD-Fc conjugate is referred to as “TMV:RBD-Fc.”FIG. 59(B) shows strong, dose-dependent binding of CR 3022 to both theRBD-Fc 121 antigen and formulated TMV to RBD-Fc 121 conjugate.Accordingly, FIG. 59 indicates the RBD-Fc 121 antigen shows greater thanfive times the reactivity to CR 3022 as compared to commercially sourcedcontrol SARS Spike and RBD reagents. This data also suggests that thepurified RBD-Fc 121 antigen maintains essential conformational epitopesin a more favorable manner compared to conventional purchased reagents.

To analyze the ability of the RBD-Fc 121 antigen to bind to the ACE-2receptor, quantitative and functional ACE-2 binding was performed usingtwo-color confocal microscopy and analysis by co-localization andcompetitive binding methods on Vero e6 cells. Vero cells are derivedfrom the kidney of an African green monkey and are commonly used incellular research. The Vero e6 cells are a subclone of Vero76,exhibiting a range of virus susceptibility. Herein, the binding abilityof the native agonist, angiotensin II, was compared with the RBD-Fcfusion by analyzing the concentration dependent ability to block bindingof an ACE-2 specific antibody to the receptor on living Vero e6 cells.Influenza H7 HA was used as a control. FAM-angiotensin II bound to Veroe6 cells as expected, with an average of a 2.79-fold increase over thenon-specific H7 control. FIG. 60 illustrates the ligand co-localizationbetween the angiotensin II and the RBD-Fc 121 antigen to Vero e6 cellsvia confocal microscopy at the following ACE-2 specific antibodyconcentrations: 10 ug/ml, 3.3 ug/ml, 1.1 ug/ml, and 0.12 ug/ml, whereinthe scale bars are equal to 15 In FIG. 60 , the general trend shows thatthe binding of the RBD-Fc 121 antigen to Vero e6 cells occurred in aconcentration-dependent manner with an increase of 2.58-fold increaseover the H7 HA control. In other words, increased binding of the RBD-Fc121 antigen (at the higher concentrations) was observed to correlatewith decreased detection of ACE-2 antibody (at the lowerconcentrations).

FIG. 61(a) also illustrates the ligand co-localization events shown inFIG. 60 . FIG. 61(b) illustrates that adding a co-localization controlreduces all specific binding, demonstrating a reduction in ACE-2antibody binding in the presence of both angiotensin II and the RBD-Fc121 antigen. As shown in FIGS. 60 and 61 , the binding of H7 HA did notimpact the detection of ACE-2 by monoclonal antibody at anyconcentration. These figures also illustrate that the RBD-Fc 121 antigeneffectively competes for ACE-2 antibody binding on Vero e6 cells andco-localizes with ACE-2 cells with similar affinity and specificity asangiotensin II.

Accordingly, FIGS. 59-61 illustrate that the RBD-Fc 121 antigenmaintains functional conformation and activity through the binding of CR3022 and ACE-2. The binding to ACE-2 shows similar affinity andspecificity as the native agonist, angiotensin II, suggesting that theconformation of the spike protein RBD in the recombinant RBD-Fc 121fusion peptide complex is comparable to that observed with native SARS-2Spike protein. This data indicates that the RBD-Fc 121 antigen displaysthe correct structure necessary to elicit the production of neutralizingantibodies against the RBD of the Covid-19 spike protein.

In Vivo Testing—Example 15(c)

The TMV NtK:RBD-fc 121 Covid-19 vaccine described above was evaluatedfor immunogenicity in two parallel evaluations using female C57BL/6mice. As shown in FIGS. 62(A)-(D), the collected serum was tested fortotal IgG anti-RBD reactivity by ELISA, using recombinant COV2 RBD-Hisas capture antigen to compare immune response between groups.

In the first evaluation, 10 animals were used per group wherein 5animals per group were sacrificed at day 14 to have sufficient sera forbroad testing. The 5 remaining animals per group were boosted on day 14and sacrificed on day 28. In the second evaluation, 5 animals were usedper group, each receiving prime and boost vaccinations on days 0 and 14respectively, with blood draws at days 0, 12, 14, 28, and terminal fromall animals. In both evaluations, the trial endpoints includemeasurement of antigen-specific geometric mean antibody titers inducedin mice, SARS-2 neutralizing titers, and antibody isotype analysis. Thetrials are outlined in Table 50 below which included various dosages,different vaccines (including purified TMV NtK only, RBD-Fc only, andthe TMV:RBD-Fc conjugate), and compared identical doses both with andwithout the use of CpG oligodeoxynucleotides (CpG) as an adjuvant. TheCpG was added to the vaccine formulation such that the concentration ofthe antigen remains the same in a consistent 100 mcL injection as theneat (non-adjuvanted) vaccine formulation. In some embodiments,monophosphoryl lipid A (MPLA) and/or SE-M were utilized as adjuvants forenhancing the immune response of the subject to the vaccine. “SE-M” is atype of stable emulsion, which typically uses a MIA Toll-Like Receptoragonist as the adjuvant.

TABLE 50 TMV NtK:RBD-Fc Vaccine Immunogenicity Testing in Mice GroupVaccine Antigen Dose (μg) Adjuvant Immunization Days Bleed Dates 1APurified TMV n/a none 0 0, 14 only 1B Purified TMV n/a none 0, 14 0, 14,28 only 2A RBD-Fc 45 none 0 0, 14 antigen only 2B RBD-Fc 45 none 0, 140, 14, 28 antigen only 3A TWA:BD-Fe 15 none 0 0, 14 Conjugate 3BTMV:RBD-Fc l5 none 0, 14 0, 14,28 Conjugate 4A TMV:RBD-Fe 45 none 0 0,12, 14, Conjugate 4B TMV:RBD-Fe 45 none 0, 14 0, 14, 28 Conjugate 5ATMV:RBD-Fc 15 CpG (50 μg) 0 0, 14 Conjugate 5B TMV:RBD-Fe 15 CpG (50 μg)0, 14 0, 14, 28 Conjugate 6A TMV:RBD-Fe 45 CpG (50 μg) 0 0, 14 Conjugate6B TMV:RBD-Fc 45 CpG (50 μg) 0, 14 0, 14, 28 Conjugate

FIG. 62(A) illustrates the immune response (ng/mL) stimulated by RBD-Fcantigen only at 12, 28, and 42 days post first vaccination. FIG. 62(B)illustrates the immune response (ng/mL) stimulated by TMV:RBD-Fcconjugate at dosages of 15 mcg and 45 neat (unadjuvanted) at 12, 28 and42 days post-first vaccination. FIG. 62(C) illustrates the immuneresponse (ng/mL) stimulated by adjuvanted TMV:RBD-Fc conjugate (15 mcg)and TMV:RBD-Fc conjugate (45 mcg+CpG) vaccines at 12, 28 and 42 dayspost-first vaccination. In FIG. 62(D), comparative IgG titersrecognizing the RBD-Fc antigen (in black) and RBD-His (in gray) areshown from sera taken at day 42. The relative ratio of reaction toRBD-His is shown as a percentage of total, RBD-Fc response, above eachstacked plot member.

Baseline serum antibody levels were very low for RBD-His and RBD-121-Fcantigen, being <100 ng/mL. Further, no significant response was measuredfor PBS vehicle control animals, and the unconjugated RBD-Fc antigenproduced a limited antibody response demonstrating that conjugated tothe TMV NtK carrier is needed to produce a robust immune response.

As shown in FIGS. 62(B) and 62(C), all TMV:RBD-Fc vaccine groups showedmeasurable antibody responses by day 12. As shown in FIGS. 62(A), 62(B)and 62(C), a dose response was observed when comparing the RBD-Fcantigen alone and the TMV:RBD-Fc Conjugate. Further, strong boosting wasobserved from day 12 to day 28 following the second vaccination. TheTMV:RBD-Fc conjugate groups showed expansion of antibody titers from day28 to day 42, whereas CpG adjuvanted vaccines showed consistent (45μg+CpG) or declining titers (15 μg+CpG). The non-adjuvanted TMV:RBD-Fcconjugate groups showed similar quantitative titers than CpG adjuvantedvaccines with all sera analyzed simultaneously against a single captureprotein.

As shown in FIG. 62(D), the relative immune response to the SARS-2 RBDwas tested, as well as the human Fe portion of the antigen to the immuneresponse. Day 42 sera were analyzed by ELISA using either the SARS-2RBD-His or the RBD-121-Fc proteins as capture agents. ELISA data wasmeasured after 1:100 dilutions of PBS/RBD-121-Fc antigen immune groups,and 1:1000 for TMV:RBD-Fc conjugate groups. Titers recognizing the RBDwere significantly lower than for RBD-Fc antigen, with the percentage ofanti-RBD varying by the TMV:RBD-Fc group from 12-35% (FIG. 62(D)), Theimmune response titers were not significantly different across theTMV:RBD-Fc conjugate groups, regardless of the capture antigen.

A favorable balance of Th1/Th2 cytokines produced facilitates a safe andeffective immune response, by balancing proinflammatory andanti-inflammatory responses Given the importance of Th1/Th2 balance, IgGisotype analysis was also conducted by measuring IgG1 (Th2) and IgG2(IgG2a+IgG2c; Th1)) isotype for individual sera taken at day 42 and theresults are shown in FIG. 63 . FIG. 63(A) illustrates the Th1 response(IgG2a and IgG2c), FIG. 63(B) illustrates the Th2 response (IgG1), andFIG. 63(C) is a stack plot comparison of relative IgG2 versus IgG1antibody by ELISA. In FIG. 63 , the “*” indicates a p<0.05 compared toother groups.

As expected, the PBS and RBD-Fc antigen groups showed low overall IgG2antibody responses, compared with all TMV:RBD-Fc conjugate groups. AllTMV:RBD-Fc conjugate groups were different from each other, withsignificantly improved IgG2 titers by dose and by addition of adjuvant.The IgG1 titers were significantly higher in non-adjuvanted TMV:RBD-Fcconjugate groups, compared with all other groups, with the CpGadjuvanted group significantly lower and equivalent to RBD-Fc alonegroup. The ratio of IgG1 to G2 isotype varied across groups, withprimarily IgG1 isotype titer in the RBD-121-Fc group, an IgG1 to G2ratio greater than 1 for non-adjuvanted TMV:RBD-fc conjugate groups, anda IgG1 to G2 ratio less than 0.1 for TMV:RBD-Fc conjugate+CpG adjuvantgroups. In summary, the CpG adjuvant strongly skewed the isotyperesponse to TMV:RBD-Fc conjugate vaccine to the Th1 type, withnon-adjuvanted TMV:RBD-Fc conjugate showing a more balanced mix ofTh1/Th2 response. The unconjugated protein stimulated almost entirely aTh2 response.

In the first evaluation mentioned above, SARS-2 neutralizing titers weremeasured using Vero E6 cell viability tests from day 14 samples. FIG. 64shows the mean cell viability after co-incubation of SARS-2 virus withmurine sera, wherein the average cell viability as percentage of totalcells is plotted against the serum dilution. The data shown in FIG. 64illustrate significantly increased viability observed in TMV:RBD-Fc 121vaccine groups (45 mcg alone, 15 and 45 mcg CpG) compared with thecontrol groups—vehicle alone and antigen alone. The geometric meanneutralization titers were calculated for each group and are shown inTable 51.

TABLE 51 Geometric Mean Neutralization Titers Induced by TMV:RBD-Fc 121Vaccine in Mice GMT Group Neutralization Titer Vehicle alone <16 Antigenalone (RBD-Fc) <16 TMV:RBD-Fc 121 <16 vaccine 15 mcg TMV:RBD-Fc 121 32vaccine 45 mcg TMV:RBD-Fc 121 35.8 vaccine 15 mcg + CpG TMV:RBD-Fc 12164 vaccine 45 mcg + CpG

The data in Table 51 show measurable neutralization titers induced inmice following prime vaccine for adjuvanted and neat (non-adjuvanted)TMV:RBD-Fc 121 vaccines.

In the second evaluation, a recombinant RBD protein (6-His fusion) wasused as the capture protein and antigen specific antibodies weremeasured and expressed as ng IgG bound/mL of pooled group sera. FIG. 65illustrates the measured response titers following vaccine 1 (prime; day12) and vaccine 2 (boost, day 28) induced in mice by TMV:RBD-Fc 121vaccines. The samples represent analysis of pooled sera from 5 animalsin each group and the capture antigen is a recombinant RBD-6-Hisprotein. As shown in FIG. 65 , measurable responses were observed at day12 (following vaccine 1—prime vaccination) for the TMV:RBD-Fc 121vaccine at 45 mcg neat or mixed with CpG adjuvant. Following vaccination2 (indicated by “boost” in FIG. 64 ) at day 28, similar titers werepresent for TMV:RBD-Fc 121 vaccine at 45 mcg neat or mixed with CpGantigen group sear. Moreover, these titers appear significantly greaterthan that induced by TMV:RBD-Fc 121 vaccine at 15 mcg neat or mixed withCpG antigen. In FIG. 65 , all TMV:RBD-Fc 121 groups are significantlyhigher than the immune response induced by RBD-Fc antigen alone. Thesetrials show dose dependency of the TMV:RBD-Fc 121 vaccine and revealthat adjuvant does not make a significant contribution to enhancedimmune responses to the RBD-Fc 121 antigen when conjugated toinactivated TMV particles. Moreover, as shown in FIG. 65 , theTMV:RBD-Fc 121 vaccine induced a level of response greater than 600mcg/mL when provided at the 45 mcg dose.

Using a SARS-2 plaque assay, the sera was tested for the generation ofvirus neutralizing antibody (Nab) titers in immunologically naïveanimals. Nab titer was detectable following a single immunization butwas greatly increased to <4000 GMT following a second (boosterimmunization). As shown in Table 52 below, two different neutralizationassay methods were used showing comparable titers and providing a highdegree of validity to the results. It should be noted that adjuvant didnot increase the Nab titer in either experiment. Data support theeffectiveness of as little as 15 mcg of TMV:RBD-Fc 121 vaccine—as itshows Nab titer of ˜200 post first dosing and >4,000 after second dosein one trial and >600 titer in second trial.

TABLE 52 Neutralizing Antibody Titers Induced by TMV:RBD-Fc 121 vaccinesin Two Murine Pre-Clinical Trials as Measured by Geometric Mean Titer(GMT) Using CPE Neutralizing Assay and PRNT50 Methods Murine Trial 2Murine Trial 1 Post- Post- Post- Post- Post- Vaccine 1: Vaccine 2:Vaccine 2: Post- Vaccine 1: Vaccine 2: Day 12** Day 28** Day 42**Vaccine 2: Day 14* Day 28* Pooled Pooled Pooled Day 42* VaccinationGroup GMT GMT PRNT50 PRNT50 PRNT50 GMT Vehicle Only <32  <64 <20 <20 <20<64 TMV + RBD-121-Fc  56 1176 <20 NA NA NA (45 mcg) RBD-121-Fc (45 mcg)NA NA <20 <20 80 294 TMV:RBD-Fc 121 194 4096 <20 20 640 676 Conjugate(15 mcg) TMV:RBD-Fc 121 (45 128 1783 <20 320 5120 2702 mcg) TMV:RBD-Fc121 (15 169 1176 <20 20 320 338 mcg) + CpG (50 mcg) TMV:RBD-Fc 121 (45388 1552 <20 320 1280 1552 mcg) + CpG (50 mcg) *TCID50 value: CPEneutralization assay **PRNT50: p < 0.05 compared with virus control

Further analysis of murine sera showed a balanced Th1/Th2 immuneresponse elicited by TMV:RBD-Fc 121 vaccines and, immunogenicity to thevaccine was heavily reliant upon conjugation to the TMV NtK carrier. Inone in vitro model to assess the potential of the TMV:RBD-Fc vaccine toinduce antibody-enhanced disease (ADE), sera containing neutralizingantibody titers of ≥2700 displayed no evidence of enhanced SARS-CoV-2entry into macrophages. This indicates the vaccine strategy stimulatesNab titers without promoting ADE.

In addition, Th1 immune responses were explored through cellularstimulation analysis. In this study, groups of 5 mice were immunized bysubcutaneous dosing once at day 1 and day 14, with either 15 μg or 45 μgantigen dose, with or without CpG, in 50 mcL, total volume (PBScomprising buffer). On days 0, 12, 18 and 42 sera were collected, andthe magnitude of the antibody response to the vaccine antigen wasassessed as total titers against the target antigen and compared withPBS immune sera or pre-immune sera. Unconjugated protein RBD-Fc was usedas the target for IgG assessment as well as RBD-HIS to assess responseto total antigen and RBD (spike S1 domain) component of antigen.Approximately 6 weeks after the first immunization, vaccinated mice wereeuthanized and terminal sera collected, and spleens harvested for IFNγELISpot analysis. An overview of the study is shown in Table 53,

TABLE 53 Overview of Matrix Study Antigen Immu- Dose nization BleedGroup Vaccine (μg) Adjuvant Days Dates 1 Vehicle Only n/a none 0, 14 0,12, 28, 42 2 RBD-Fc 45 none 0, 14 0, 12, 28, 42 3 TMV:RBD-Fc 15 none 0,14 0, 12, 28, 42 4 TMV:RBD-Fc 45 none 0, 14 0, 12, 28, 42 5 TMV:RBD-Fc15 CpG 0, 14 0, 12, 28, 42 (50 μg) 6 TMV:RBD-Fc 45 CpG 0, 14 0, 12, 28,42 (50 μg)

Spleens from two animals of each group were harvested at day 42, singlecell suspensions were generated, cells were stimulated for 36 hours withthe SARS-CoV-2 RBD-HIS protein and the number of IFNγ secreting cellswere measured. The results are shown in FIG. 66 , wherein FIG. 66(A)illustrates the IFNγ ELISpot analysis for vehicle and RBD-Fc only, FIG.66(B) illustrates the IFNγ ELISpot analysis for unadjuvanted TMV:RBD-Fcvaccine, and FIG. 66(C) illustrates the IFNγ ELISpot analysis forTMV:RBD-Fc vaccine with the CpG adjuvant.

As shown in FIG. 66 , only cells recovered from mice vaccinated withTMV:RBD-Fc vaccines containing the CpG adjuvant induced statisticallysignificant numbers of IFNγ secreting cells at day 42 (four weekspost-2nd immunization), with equivalent numbers in both 15 and 45 lagdose groups (FIG. 66(C)). This data is supportive of the IgG isotypeanalysis which indicates that TMV:RBD-Fc vaccine with CpG adjuvantco-delivery stimulates Th1 responses in mice.

In addition, a VaxArray® SeroAssay analysis of murine sera was conductedfrom mice immunized with various TMV:RBD-Fc vaccine dosages, and bothwith and without adjuvant, to evaluate the relevance of binding toheterologous coronavirus antigens. In FIG. 67 , nCoV antigen (i) is fullSARS-CoV2 spike protein, (ii) is the S1 (RBD) domain, SARS(i) is fullSARS-1 spike as is MFRS(i) and HKU1(i), each produced in mammaliansystems. The relative titer is represented as fold enhancement of signalover controls reported. Strong reactivity was noted to both SARS-2antigens and apparent cross reactivity to SARS-1 antigen. As shown inFIG. 67 , no binding was observed to divergent MERS or HKU1 spikeproteins. Accordingly, the results in FIG. 67 indicate that anti-serafrom mice with neutralization titers recognized heterologously produced(mammalian cell) SARS-2 Spike proteins, RBD, and SARS-1 Spike Proteins(i.e. exhibit cross reactivity).

The murine testing also studied a murine macrophage line (Raw 264.7)lacking ACE-2 binding domains, which found that anti-spike RBDneutralizes viral killing and does not promote antibody-dependentenhancement of infection in murine macrophages. In humans, ACE-2functions as a cell surface receptor for the spike protein of SARS-CoV2during the invasion of respiratory epithelial cells. Concomitantly, inhumans, mice and other organisms, neutralizing antibodies against thereceptor binding domain of this spike protein have been found inpatients that have recovered from SARS-CoV and are believed to beprotective against infection with SARS-CoV2. However, it has also beenshown that non-neutralizing antibodies are able to enhance the severityof SARS-CoV infections, and have impeded successful vaccine developmentfor coronaviruses, including SARS-CoV1 and SARS-CoV2.

In the murine testing study, the abilities of certain antibodiesincluding ones generated by the subject TMV:RBD-Fc vaccines werecompared in regards to antibody-dependent enhancement of SARS-CoV2infection. Raw 264.7 cells were grown into a 96 well plate in VGCM andallowed to adhere overnight. Cells were washed extensively prior to theaddition of sera or antibodies. The antibodies of the study (or pooledsera) were simultaneously incubated for 1 hour to facilitate viralinactivation by neutralizing antibodies prior to incubation withmacrophages. Following this incubation, media containing both antibodiesand SARS-CoV2 was added to the macrophages and incubated for 48 hours,at which time viability was assessed thru the addition of Cell Titer Glo(Promega) and evaluated on the basis of luminescence output.

As further shown in FIGS. 68 (A-B), non-neutralizing antibodies againstthe nucleocapsid and spike proteins of SARS-CoV2 enhanced viral entryinto murine macrophages, a cell type that does not express ACE2 on thecell surface and is normally resistant to infection. Viability ofmacrophages exposed to the virus in the absence of antibody averaged91.5% of the cell viability control at 48 hours post-infection (n=10).FIG. 68(A); FIG. 68(B), first and fifth bars. In the presence ofanti-nucleocapsid antibodies raised against the whole spike protein at6.25 ug/ml, viability fell to an average of 55.69% with the greatestconcentration-dependent drop above 1 ug/ml. FIG. 68(A). In the presenceof polyclonal antibodies raised against the whole spike protein at 6.25ug/ml, viability fell to an average of 59.78%. FIG. 68(A). In contrast,pooled murine sera from the study in Example 15(b) against TAP COVID-19vaccine at 6.25 ug/ml showed high neutralization titers withoutobservable antibody-dependent enhancement, as indicated by an averageviability of 125.56% compared to the cell viability control. Statisticaldifferences were measured using one-way ANOVA with Tukey's P values,with the results provided in FIG. 68B. Accordingly, non-neutralizingantibodies (against the nucleocapsid or spike protein) promoted viralentry into the macrophages, while antibodies generated from theTMV:RBD-fc vaccines did not demonstrate this effect.

Accordingly, the in vivo testing shows that TMV:RBD-Fc 121 vaccinesinduce measurable and statistically significant SARS-2 neutralizingantibody titers over controls. The TMV:RBD-Fc 121 vaccine of the presentexample shows a strong, vigorous, and promising immune response. Itshould also be noted that the entire vaccine manufacturing process, fromthe initial sequencing of the Covid-19 antigen to the final productionof the cGMP vaccine for drug product sterility and release, can beaccomplished in 8 weeks of time, which is a significant advantage overconventional vaccine production methods which typically take more than 6months.

Furthermore, as described in Example 14, the studies with the QIVvaccine show very high promise in experimental animals for H1, H3, H5,and H7 influenza models, and no toxicologically significant findingswere observed. This data, as well as the role of inactivated TMV as acarrier, suggests similar potential for the Covid-19 vaccines disclosedherein due to the similar structural components and developmentplatforms. Based on the stability of the QIV vaccine discussed inExample 13, it is likewise expected that the Covid-19 vaccines disclosedherein will exhibit high stability at room temperature for at least asix month period.

Accordingly, through the practice of certain embodiments herein, aconjugable antigen can be manufactured, suitable for conjugation acarrier. The teachings herein contemplate and are otherwise directed toantigens which can be conjugated to virus particles, virus particles,vaccines, constructs, and other compositions of matter as well as allmethods employed in the making of these. Such an antigen may be arecombinant antigen, and generally comprises a fusion peptide having afirst peptide which comprises a receptor binding domain of a pathogen, anon-limiting example being a coronavirus, and a second peptide whichcomprises a fragment crystallizable (Fe) region of an antibody capableof binding to a Fe receptor. In some embodiments, the first peptide andthe second peptide are linked by a hinge portion. Optionally, this hingeportion may be a portion of the Fe region. In some embodiments, thehinge portion contains an amino acid sequence as set forth in SEQ ID NO:3. Exemplary coronaviruses from which a receptor binding domain may beobtained include without limitation SARS-CoV-1, SARS-CoV-2, and MERS.

Now with reference to FIG. 50 , an exemplary receptor binding domain ofa SARS-CoV-2, the receptor binding domain is contained in a S-1 subunitof a spike protein of the coronavirus, and it comprises contact residueswhich are located in a range from about position 289 to about position662. In some embodiments, the second peptide may comprise an Fe domainof an IgG1 antibody, with such domain containing as noted herein anamino acid sequence as set forth in SEQ ID NO: 4.

Consistent with the teachings herein, in some embodiments a vaccine ismanufactured, comprising at least one conjugable antigen, as describedaccording to the multiple embodiments and alternatives of thisdisclosure, and a carrier comprising a virus particle. In someembodiments, such a virus particle is a virus, for example TMV. Thefirst peptide of the conjugable antigen may contain an amino acidsequence as set forth in SEQ ID NO: 2, alternatively the amino acidsequence may be as set forth in SEQ ID NO: 8. The first peptide maycontain contact residues located in a range from about position 289 toabout position 662 of a S-1 subunit of a spike protein of thecoronavirus, which will contact an ACE-2 receptor on a cell of amammalian subject to whom such a vaccine is administered, after theantigen is released from the virus particle carrier followingadministration. For the RBD-FC 121 antigen, the contact residues may belocated in a range from about position 301 to about position 662 of theS-1 subunit. It has been found that with regard to the conjugableantigen referred to herein as the RBD-FC 121 antigen, the first peptidelacks an amino acid sequence as set forth in SEQ ID NO: 5, while thefirst peptide of RBD-Fc 139 antigen does contain this sequence. In bothcases, when the conjugable antigen of the vaccine is released in amammalian subject, having cells that include one or more ACE-2receptors, the at least one conjugable antigen binds to the one or moreACE-2 receptors. In some embodiments, a virus particle used in themanufacture of such a vaccine has surface lysine residues, whichchemically associate with the fusion peptide, more specifically thefirst peptide, resulting from the conjugation reaction.

Consistent with the breadth of teachings herein, in some embodiments avaccine in accordance with teachings herein is multivalent and comprisesat least one conjugable antigen having a receptor binding domain of atype A influenza virus and at least one conjugable antigen having areceptor binding domain of a coronavirus. Other possible combinationsare within the scope of present embodiments, and the combinationsprovided herein are illustrative and non-limiting. In some embodiments,a vaccine in accordance with teachings herein is multivalent andcomprises at least one conjugable antigen having a receptor bindingdomain of a type B influenza virus and at least one conjugable antigenhaving a receptor binding domain of a coronavirus. Alternatively, avaccine in accordance with teachings herein is multivalent and comprisesat least one conjugable antigen having a receptor binding domain of atype A influenza virus, at least one conjugable antigen having areceptor binding domain of a type B influenza virus, and at least oneconjugable antigen having a receptor binding domain of a coronavirus.Various ratios may be selected and expressed as follows, virusparticle:antigen by wt (i.e., ratio of the virus particle and the atleast one conjugable antigen by weight). As desired, this ratio may bein a range between about 1:1 and about 8:1, more specifically 8:1.Optionally, CpG may be included with a vaccine in accordance with theteachings herein as an adjuvant for enhancing the immune response of themammalian subject to the vaccine.

Example 16—Coronavirus Vaccine Candidate: Plant Expression,Purification, Characterization, and Conjugation of RBD-Fc 139 (SARS-2)to TMV

In addition to the RBD-Fc 121 antigen, it is expected that any type ofvirus antigen, including other Covid-19 antigens, may be purified andconjugated to inactivated TMV for use as an effective vaccine candidateaccording to multiple embodiments and alternatives. In some embodiments,the following antigen sequence (referred to herein as “RED-Fc 139Construct”) is used for the synthesis of a Covid-19 antigen:

1. “(SEQ ID NO: 1”) Signal Peptide: MGKMASLFATFLVVLVSLSLASESSA2. “(SEQ ID NO: 5”) SARS-2 virus Spike amino acids numbered 319-330:RVQPTESIVRFP 3. (SEQ ID NO: 6”) SARS-2 virus Spike aminoacids numbered 331-591: NITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDITPCS4. “(SEQ ID NO: 7”) Tobacco etch virus NIA cleavage sequence: enlyfqg5. “(SEQ ID NO: 3”) Fc Hinge: VEPKSCDKTHTCPPCP6 “(SEQ ID NO: 4”) IgG1 171 allotype Fc:APELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTCLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVFSCSVMHEALHNHYTQKSLSLSPG

In an embodiment of the RBD-FC 139 Construct, the following antigensequence is used for the synthesis of a Covid-19 antigen:

“(SEQ ID NO: 8”) SARS-2 virus Spike amino acids numbered 319-591:RVQPTESIVRFPNITNLCPFGEVFNATRFASVYAWNRKRISNCVADYSVLYNSASFSTFKCYGVSPTKLNDLCFTNVYADSFVIRGDEVRQIAPGQTGKIADYNYKLPDDFTGCVIAWNSNNLDSKVGGNYNYLYRLFRKSNLKPFERDISTEIYQAGSTPCNGVEGFNCYFPLQSYGFQPTNGVGYQPYRVVVLSFELLHAPATVCGPKKSTNLVKNKCVNFNFNGLTGTGVLTESNKKFLPFQQFGRDIADTTDAVRDPQTLEILDIT PCS

Multigenic constructs were designed and built to contain genes encodingthe proteins necessary to synthesize the RBD-Fc 139 antigen, which alsotargets the RBD domain of SARS-2 in a similar manner to RBD-Fc 121.According to multiple embodiments and alternatives, the RBD-Fc 139construct was ligated into the TRBO vector (as a non-limiting example)and subsequent colonies were screened to confirm clones. The assembledexpression plasmids containing the RBD-Fc 139 Construct, similar to theplasmid shown in FIG. 51 , were amplified, and then purified for use inthe production of a Master Cell Bank.

According to multiple embodiments and alternatives, the RBD-Fc 139antigen is expressed in plants and purified using an antigenpurification platform described herein. As shown in FIGS. 69-70 , theantigen purification platform, according to multiple embodiments andalternatives, successfully purified RBD-Fc 139 resulting in high yieldswith pure, stable antigen as RBD-Fc monomer in a manner that iscompliant with GLP regulations. FIG. 69 , taken from the conclusion ofthe antigen purification platform, contains a SDS page gel indicatingpurity and successful purification for the RBD-Fc 139 antigen in bothreducing and non-reducing conditions. In FIG. 69 , the lanes include:lane 1—11.8 ug of purified antigen in reducing conditions, lane 2—blank,lanes 3-5:1.5 ug of purified antigen in reducing conditions, lane6—blank, lane 7—marker, lane 8—blank, lanes 9-11—1.5 ug of purifiedantigen in non-reducing conditions, lane 12—blank, and lane 13, 11.8 ugof purified antigen in non-reducing conditions. The clear and visiblebands indicate the RBD-Fc 139 antigen product is highly pure.

FIG. 70 is the SEC-HPLC report of the free RBD-Fc 139 antigen, whichproduced the signal detailed in Table 54 below. The 100% area under thepeak indicates that 100% of the RBD-Fe 139 antigen is in monomeric form.

TABLE 54 SEC-HPLC Data of free, purified RBD-Fc 139 Antigen RT WidthArea Peak [min] [min] Area Height % Symmetry 17.110 1.18 2220.84 23.81100 0.78

Therefore, the SDS page gel and the SEC-HPLC report of the free RBD-fc139 antigen confirm the antigen purification platform which was used,according to multiple embodiments and alternatives, successfullypurified the RBD-Fc 139 antigen.

In parallel to the Covid-19 antigen production, the TMV NtK virions withsurface lysine residues for efficient conjugation are manufactured inplants and purified according to the virus purification platformdescribed herein. Following purification, the TMV Ntk is subject tomicron filtration and immediately treated with UV inactivation. TheRBD-Fc 139 antigen is then conjugated to the inactivated TMV via thesurface exposed lysine residues utilizing the conjugation of recombinantantigen embodiments described herein.

Also in accordance with the present embodiments herein, a TMV:RBD-Fc 139conjugate is currently being studied as a vaccine candidate for Covid-19Disease. However, based on the success of the quadrivalent vaccine andthe strong immune responses by the initial TMV:RBF-Fc 121 conjugate, theTMV:RBD-Fc 139 conjugate is also expected to be a viable Covid-19vaccine.

It will be readily appreciated that the breadth of teachings hereinaccords with multiple embodiments with a broad array of options inalternative manners for practicing the embodiments. Accordingly, andwithout limitation, in an embodiment, referred to herein as embodimentA, directed to an antigen, a fusion peptide is formed of a first peptidewhich comprises a receptor binding domain of a pathogen, and a secondpeptide which comprises a fragment crystallizable (Fe) region of anantibody capable of binding to a Fe receptor. In an embodiment withinthe scope of embodiment A, and referred to herein as embodiment B, theantigen further comprises a hinge portion linking the first peptide andthe second peptide. Being a portion of the Fc region and morespecifically as referred to herein as embodiment C, the hinge portionmay contain an amino acid sequence as set forth in SEQ ID NO: 3. In anembodiment within the scope of embodiment A and referred to herein asembodiment D, the pathogen is a coronavirus having a receptor bindingdomain, and more specifically as referred to herein as embodiment E, thecoronavirus is chosen from the group consisting of SARS-CoV-1 andSARS-CoV-2. In an embodiment within the scope of embodiment E, andreferred to herein as embodiment F, the receptor binding domain of thecoronavirus comprises contact residues are located in a range from aboutposition 289 to about position 662 of a S-1 subunit of a spike proteinof the coronavirus, wherein the contact residues contact an ACE-2receptor on a cell of a mammalian subject, or alternatively, in anembodiment referred to herein as embodiment G, the contact residues arelocated in a range from about position 301 to about position 662 of theS-1 subunit. In an embodiment within the scope of embodiment F, andreferred to herein as embodiment H, the receptor binding domain of thecoronavirus lacks an amino acid sequence as set forth in SEQ ID NO: 5.

In an embodiment within the scope of embodiment D, and referred toherein as embodiment I, the coronavirus is a Middle East respiratorysyndrome coronavirus. In an embodiment within the scope of embodiment A,and referred to herein as embodiment J, the second peptide is an Fcdomain of an IgG1 antibody and more specifically as referred to hereinas embodiment K, the Fc domain contains an amino acid sequence as setforth in SEQ ID NO: 4 and the first peptide contains an amino acidsequence as set forth in either SEQ ID NO: 2 or SEQ ID NO: 8.Accordingly, an antigen may be practiced (i.e., made, formed, designed,used, and so forth) in accordance with embodiment A as more fullydescribed herein. Optionally and as desired by those practicing theembodiments herein, an antigen as described herein may be practiced byincorporating with embodiment A any one or more of embodiments B, C, E,F, G, H, I, or J, and embodiments may be directed to compounds, methods,and genetic constructs in the practice of any one or more of thesealternative embodiments. Likewise, embodiments may be directed to avaccine comprising an antigen, a method of forming an antigen, or agenetic construct useful in forming an antigen, as recited in embodimentA, B, C, D, E, F, G, H, I, or J, and further comprising in combinationwith any aforementioned embodiment, a carrier comprising a virusparticle, wherein the virus in some embodiments is a virus, and moreparticularly a tobacco mosaic virus, and wherein the fusion peptidechemically associates with lysine residues on a surface of the earlier.

Still further, and without limitation, in an embodiment referred toherein as embodiment K, a vaccine comprises an influenza hemagglutininantigen (HA) and a carrier comprising a virus particle having surfacelysine residues, wherein the HA chemically associates with the surfacelysine residues. In an embodiment within the scope of embodiment K, andreferred to herein as embodiment L, the virus particle releases the atleast one antigen in a mammalian subject having cells that include oneor more ACE-2 receptors, the at least one antigen binds to the one ormore ACE-2 receptor. In an embodiment within the scope of embodiment K,and referred to herein as embodiment M, the virus particle is a virus,or more specifically as referred to herein as embodiment N, the virus isa tobacco mosaic virus. In an embodiment within the scope of any ofembodiment K, L, M, or N, and referred to herein as embodiment O, thevaccine is multivalent, and the HA is chosen from the group consistingof type A HA and type B HA, and the vaccine further comprises at leastone antigen having a receptor binding domain of a coronavirus, whereinthe at least one antigen having a receptor binding domain of acoronavirus chemically associates with the surface lysine residues. Inan embodiment within the scope of embodiment O, and referred to hereinas embodiment P, the HA comprises two or more type A hemagglutininantigens (HAs) and two or more type B HAs. Also, in an embodiment withinthe scope of embodiment O, and referred to herein as embodiment Q,additional features found in any one or more of embodiments A, B, C, D,E, F, G, H, or J are incorporated with the coronavirus element of thevaccine. Further, in an embodiment referred to herein as embodiment R,in a vaccine within the scope of embodiment Q, a ratio of virus particleand at least one antigen (by weight) is in a range between 1:1 and 8:1and, more particularly in an embodiment referred to as embodiment S thatrange is 8:1.

Additional Embodiments and Uses of Novel Subject Matter Herein

In addition to antigens described above, including the RBD-Fc antigensfor treatment against SARS-CoV2, myriad other antigens can be formedaccording to the multiple embodiments and alternatives described herein.The scope of the descriptions and teachings herein are intended to belimited only in accordance with the claims. For example, a strategysimilar to Examples 14, 15, and 16 can be employed to derive candidatevaccines from other human-infecting coronaviruses, including acuterespiratory syndrome coronavirus (SARS-1) and Middle East respiratorysyndrome coronavirus (MERS). As illustrated in FIG. 71 , domains ofhomology can be identified into functional regions in the Spike S1domain which correlated with receptor binding and other essentialactivities.

In this regard, FIG. 71 shows the Receptor Binding Domain of the spikeprotein sequence alignment of SARS-CoV-2 and other relatedCoronaviruses. Shown here is a sequence alignment for the interacting(i.e. binding) domain of SARS-CoV-2 (MN938384), Bat-CoV (MN996532 andMG772933) and SARS-CoV (NC004718). The key amino acids described for theinteraction with ACE2 and SARS-CoV2 are underlined. (Lines (-)=sameamino acid, dots (.)=deletion). FIG. 71 is from Ortega J T, Serrano M L,Pujol F H, Rangel H R. Role of changes in SARS-CoV-2 spike protein inthe interaction with the human ACE-2 receptor: An in silico analysis.EXCLI J. 2020; 19:410-417. Published 2020 Mar. 18.Doi:10.17179/excli2020-1167.

As seen in the constructs for the RBD-Fc 121 antigen and RBD-Fc 139antigen, and reading the N-terminus, the core elements of functionalityextend from the “RVQPT” motif for the RBD-Fc 139 Construct to the“CGPKK” domain for both the RBD-Fc 121 and RBD-Fc 139 Constructs.Looking more closely at the RBD-Fc 121 Constructs, there extends beyondthe core domain a more extended protein domain which facilitatesappropriate folding. Indeed, this strategy is expected to extend to anytype of coronavirus antigen, through the processes of:

-   -   1. Protein homology analysis—shown in FIG. 71 ,    -   2. In silico protein folding using extant coronavirus Spike        models,    -   3. Creation of extensin signal peptide—RBD genetic fusion that        promotes efficient cleavage as judged by SignalIP or Phobius;    -   4. Genetic fusion with Fe reading frame—as illustrated by the        RBD-Fc 121 and 139 Constructs;    -   5. Expression in plants,    -   6. Purification by Protein A and other methods illustrated in        this patent application,    -   7. Conjugation to TMV in accordance with multiple embodiments        and alternatives described herein

Such a vaccine can be formed to contain a number of different anddiverse antigens, and used to prevent an identified coronavirus pathogensuch as SARS-1 or MFRS, or formulated with either the RBD-Fc 121 or 139antigen into a multivalent vaccine to prevent SARS-1, SARS-2, and HERS,as non-limiting examples. In accordance with multiple embodiments andalternatives described herein, for example with influenza A and Bantigens, multivalent TMV conjugate vaccines can be mixed as equal orproportional quantities of each TMV-antigen conjugate and applied in asingle immunization. As described herein, multivalent TMV-conjugatevaccines do not show immune dominance of one antigen preventing responseto a second or third or fourth. Indeed, both HAI and neutralizingantibodies were generated against all strains in animals immunized witha quadrivalent TMV influenza vaccine, as seen with Example 14. Usingthis quadrivalent vaccine, protection can be measured against more thanone individual influenza vaccine.

Thus, it is anticipated that the approaches described herein, inaccordance with multiple embodiments and alternatives, are likely toprovide a wide and varied range of antigens upon a single virus particlecarrier. To illustrate in a non-limiting way, in an embodiment amultivalent vaccine is provided in accordance with the teachings herein,comprising two, three, four, five, or more different antigens againstvarious viruses and other pathogens. At least one antigen may neutralizeor stimulate an immune response against one type of virus (e.g.,influenza), while at least one other antigen may do the same againstanother type of virus (e.g., a pandemic virus like coronavirus). Anantigen occupying a position on the carrier could be comprised of afusion protein modeled after the receptor binding domain of a virus,combined with a fusion partner, such as the Fc domain of an antibodyfrom the tail region of an IgG1 molecule. The influenza portion of theat least one antigen on a single vaccine may comprise both type A andtype B influenza. Likewise, an approach may provide at least one antigendirected against one or more coronaviruses. The flexibility of theapproaches herein and wide scope of combinable antigen-virus conjugatescontemplated herein promote the ability to manufacture broad spectrumvaccines at a large scale in a compressed time period, often measuringweeks as opposed to many months.

It will be understood that the embodiments described herein are notlimited in their application to the details of the teachings anddescriptions set forth, or as illustrated in the accompanying figures.Rather, it will be understood that the present embodiments andalternatives, as described and claimed herein, are capable of beingpracticed or carried out in various ways. Also, it is to be understoodthat words and phrases used herein are for the purpose of descriptionand should not be regarded as limiting. The use herein of “including,”“comprising,” “e.g.,” “containing” or “having” and variations of thosewords is meant to encompass the items listed thereafter, and equivalentsof those, as well as additional items.

Accordingly, the foregoing descriptions of several embodiments andalternatives are meant to illustrate, rather than to serve as limits onthe scope of what has been disclosed herein. The descriptions herein arenot intended to be exhaustive, nor are they meant to limit theunderstanding of the embodiments to the precise forms disclosed. It willbe understood by those having ordinary skill in the art thatmodifications and variations of these embodiments are reasonablypossible in light of the above teachings and descriptions.

What is claimed is:
 1. An antigen, comprising: a fusion peptide having afirst peptide which comprises a receptor binding domain of a pathogen,and a second peptide wherein the fusion peptide is capable of beingchemically linked with a virus particle, and wherein the second peptideis a fragment crystallizable (Fc) region of an antibody capable ofbinding to a Fc receptor.
 2. The antigen of claim 1, wherein the Fcregion is an Fc domain of an IgG1 antibody.
 3. The antigen of claim 2,wherein the Fc domain contains an amino acid sequence as set forth inSEQ ID NO:
 4. 4. The antigen of claim 1, further comprising a hingeportion linking the first peptide and the second peptide.
 5. The antigenof claim 4, wherein the hinge portion is a portion of the Fc region ofsaid antibody.
 6. The antigen of claim 5, wherein the hinge portioncontains an amino acid sequence as set forth in SEQ ID NO:
 3. 7. Theantigen of claim 1, wherein the pathogen is a coronavirus having saidreceptor binding domain.
 8. The antigen of claim 7, wherein thecoronavirus is chosen from the group consisting of SARS-CoV-1 andSARS-CoV-2.
 9. The antigen of claim 8, wherein the receptor bindingdomain of the coronavirus is located on a S-1 subunit of a spike proteinof the coronavirus and comprises contact residues located in a rangefrom about position 289 to about position 662, wherein the contactresidues contact an ACE-2 receptor on a cell of a mammalian subject. 10.The antigen of claim 9, wherein the contact residues are located in arange from about position 301 to about position 662 of the S-1 subunit.11. The antigen of claim 9, wherein the receptor binding domain of thecoronavirus lacks an amino acid sequence as set forth in SEQ ID NO: 5.12. The antigen of claim 7, wherein the coronavirus is a Middle Eastrespiratory syndrome coronavirus.
 13. The antigen of claim 1, whereinthe fusion peptide chemically associates with surface lysine residues ofa carrier, the carrier comprising a virus particle having the surfacelysine residues.
 14. The antigen of claim 1, wherein the first peptidecontains an amino acid sequence as set forth in SEQ ID NO:
 2. 15. Theantigen of claim 1, wherein the first peptide contains an amino acidsequence as set forth in SEQ ID NO:
 8. 16. The antigen of claim 1,wherein the antigen is a recombinant antigen.
 17. The antigen of claim1, wherein the virus particle is a virus.
 18. The antigen of claim 17,wherein the virus is a tobacco mosaic virus.
 19. A vaccine, comprisingat least one antigen as recited in claim 1 and a carrier comprising avirus particle, wherein the at least one antigen is chemically linked tosaid virus particle.
 20. The vaccine of claim 19, wherein the pathogenof the at least one antigen is a coronavirus having said receptorbinding domain.
 21. The vaccine of claim 19, wherein when the virusparticle releases the at least one antigen in a mammalian subject havingcells that include one or more ACE-2 receptors, the at least one antigenbinds to the one or more ACE-2 receptors.
 22. The vaccine of claim 21,wherein the pathogen of the antigen is a coronavirus chosen from thegroup consisting of SARS-CoV-1 and SARS-CoV-2.
 23. The vaccine of claim22, wherein the receptor binding domain of the coronavirus is located ona S-1 subunit of a spike protein of the coronavirus and comprisescontact residues located in a range from about position 289 to aboutposition 662, wherein the contact residues contact an ACE-2 receptor ona cell of a mammalian subject.
 24. The vaccine of claim 23, wherein thecontact residues are located in a range from about position 301 to aboutposition 662 of the S-1 subunit.
 25. The vaccine of claim 23, whereinthe receptor binding domain of the coronavirus lacks an amino acidsequence as set forth in SEQ ID NO:
 5. 26. The vaccine of claim 19,wherein the Fc region is an Fc domain of an IgG1 antibody.
 27. Thevaccine of claim 19, wherein the antigen further comprises a hingeportion linking the first peptide and the second peptide.
 28. Thevaccine of claim 27, wherein the hinge portion is a portion of the Fcregion of said antibody.
 29. The vaccine of claim 19, wherein the virusparticle is a virus.
 30. The vaccine of claim 29, wherein the virus is atobacco mosaic virus.
 31. The vaccine of claim 19, wherein a ratio ofthe virus particle and the at least one antigen is expressed as virusparticle:antigen by wt, and said ratio is in a range between 1:1 and8:1.
 32. The vaccine of claim 19, further comprising an adjuvant forenhancing an immune response of a mammalian subject to the vaccine,wherein the adjuvant is chosen from the group consisting of CpG, MPLA,and SE-M.
 33. The vaccine of claim 31, wherein the ratio of the virusparticle and the at least one antigen is about 8:1.
 34. The antigen ofclaim 1, wherein the pathogen is a coronavirus having said receptorbinding domain, wherein said receptor binding domain of the coronaviruslacks an amino acid sequence as set forth in SEQ ID NO:
 5. 35. Thevaccine of claim 19, wherein the pathogen of the at least one antigen isa coronavirus having said receptor binding domain, wherein said receptorbinding domain of the coronavirus lacks an amino acid sequence as setforth in SEQ ID NO: 5.